Candida albicans proteins associated with virulence and hyphal formation and uses thereof

ABSTRACT

The present invention relates to  Candida albicans  proteins, such as CaCla4p, Cst20p, CaCdc42p and CaBem1p, associated with virulence and hyphal formation and uses thereof, such as to design screening tests for inhibitors for the treatment of pathogenic fungi infections and/or inflammation conditions. The invention also relates to an in vitro screening test for compounds to inhibit the biological activity of at least one protein selected from the group consisting of CaCla4p, Cst20p, CaCdc42p and CaBem1p, which comprises: a) at least one of said proteins; and b) means to monitor the biological activity of said at least one protein; thereby compounds are tested for their inhibiting potential.

[0001] This application is a continuation-in-part of U.S. Ser. No. 09/301,132 filed Apr. 28, 1999, which is a continuation of PCT/CA97/00809 filed Oct. 29, 1997 designating the United States and claiming priority from U.S. provisional patent application Ser. No. 60/029,458 filed Oct. 30, 1996.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates to Candida albicans proteins, such as CaCla4p, Cst20p, CaCdc42p and CaBem1p, associated with virulence and hyphal formation and uses thereof, such as to design screening tests for inhibitors for the treatment of pathogenic fungi infections and/or inflammation conditions.

[0004] 2. (b) Description of Prior Art

[0005]Candida albicans is the major fungal pathogen in humans, causing various forms of candidiasis. The incidence of infections is increasing in immunocompromised patients. This fungus is diploid and is capable of a morphological transition from a unicellular budding yeast to a filamentous form. Extensive filamentous growth leads to the formation of a mycelium displaying hyphae with branches and lateral buds. In view of the observation that hyphae seem to adhere to and invade host tissues more readily than does the yeast form, the switch from the yeast to the filamentous form probably contributes to the virulence of this organism (for a review see Fidel, P. L. & Sobel, J. D. (1994) Trends Microbiol. 2, 202-205). The molecular mechanisms by which morphological switching is regulated are poorly understood (Whiteway review 2000, Curr. Op. Microbio., 3:582-588).

[0006] Like C. albicans, bakers yeast Saccharomyces cerevisiae is also a dimorphic organism capable of switching under certain nutritional conditions from a budding yeast to a filamentous form. Under the control of nutritional signals, diploid cells switch to pseudohyphal growth (Gimeno, C. J. et al. (1992) Cell 68, 1077-1090), and haploid cells to invasive growth (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985).

[0007] The similarities between the dimorphic switching of S. cerevisiae and C. albicans suggest that these morphological pathways may be regulated by similar mechanisms in both organisms. In S. cerevisiae, morphological transitions are controlled by signaling components that are also involved in the mating response of haploid cells (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985; Liu, H. et al. (1993) Science 262, 1741-1744). The switch to pseudohyphal growth requires a transcription factor encoded by the STE12 gene, and a mitogen-activated protein (MAP) kinase cascade including Ste7p (a homolog of MAP kinase kinase or MEK), Ste11p (a MEK kinase homolog) and Ste20p (a MEK kinase kinase) (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985; Liu, H. et al. (1993) Science 262, 1741-1744). The MAP kinases involved in this response are as yet unknown (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985; Liu, H. et al. (1993) Science 262, 1741-1744).

[0008] Members of the Ste20p family of serine/threonine protein kinases are thought to be involved in triggering morphogenetic processes in response to external signals in organisms ranging from yeast to mammalian cells. Two of these kinases, Ste20p and Cla4p, are well characterized in S. cerevisiae (Leberer, E. et al. (1992) EMBO J. 11, 4815-4824; Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830). Ste20p is required for pheromone signal transduction (Leberer, E. et al. (1992) EMBO J. 11, 4815-4824) and for filamentous growth in response to nitrogen starvation (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985; Liu, H. et al. (1993) Science 262, 1741-1744), and shares an essential function with Cla4p during budding (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830). Ste20p and Cla4p interact with the small G-protein Cdc42p, and this interaction is required for viability of S. cerevisiae cells. Ste20p also interacts with the SH3 domain protein Bem1p, and this interaction plays a role in morphogenetic processes (Leeuw, T. et al. (1995) Science 270, 1210-1213).

[0009] Given the increase in the incidence of candidiasis, particularly in immunocompromised patients, it would be desirable to develop a means to screen and identify potential inhibitors of C. albicans.

SUMMARY OF THE INVENTION

[0010] It has now been found that Cst20p, a C. albicans homolog of the Ste20p protein kinase, is required for hyphal growth of C. albicans under certain in vitro conditions. Cst20p has also been shown to play a role in virulence, as judged from significantly prolonged survival of mice infected with CST20 deleted cells. Cst20p, thus, appears to act in a regulatory pathway that is involved in hyphal growth of C. albicans.

[0011] It has also been found that CaCla4p, a C. albicans homolog of the Cla4p protein kinase, is required for hyphal formation in vitro in response to serum, and in vivo in a mouse model for systemic candidiasis. CaCla4p is required for efficient colonization of kidneys with C. albicans cells after infection of mice and essential for virulence in the mouse model.

[0012] Accordingly, in one aspect of the present invention, there is provided isolated Candida albicans proteins, such as CaCla4p, Cst20p, CaCdc42p and CaBem1p, identified by amino acid sequences as well as by the nucleotide sequences encoding them.

[0013] In another aspect of the present invention, there is provided an in vitro screening test useful to identify potential anti-fungal compounds. The assay comprises the steps of:

[0014] a) combining at least one protein selected from the group consisting of CaCla4p (SEQ ID NO:8), Cst20p (SEQ ID NO:6), CaCdc42p (SEQ ID NO:10) and CaBem1p (SEQ ID NO:12) with a target that interacts with said protein in a protein/target interaction;

[0015] b) adding a test compound to the protein/target mixture; and

[0016] c) measuring the protein/target interaction, wherein a lack of protein/target interaction indicates that said compound is a potential anti-fungal compound.

[0017] In another aspect, the in vitro screening assay may further comprise the step of:

[0018] d) comparing the protein/target interaction in the presence or absence of a test compound, wherein a reduced protein/target interaction in the presence of a test compound indicates that said test compound is a potential anti-fungal compound.

[0019] In accordance with another embodiment of the present invention, an assay is provided by which inhibitors of the interactions between CaCla4p and CaCdc42p can be identified, as well as inhibitors of the interactions between Cst20p and CaCdc42p.

[0020] The term “fungi” when used herein is intended to mean any fungi, pathogenic or not, which show hyphal induction using kinases, such as C. albicans, Saccharomyces cerevisiae, Aspergillus, Ustilago maydis, and all the species of the fungal genera Aspergillus, Blastomyces, Candida, Cladosporium, Coccidioides, Cryptococcus, Epidermophyton, Exophilia, Fonsecaea, Histoplasma, Madurella, Malassezia, Microsporum, Paracoccidioides, Penicillium, Phaeoannellomyces, Phialophora, Scedosporium, Sporothrix, Torulopsis, Trichophyton, Trichosporon, Ustilago, Wangiella, Xylohypha, among others.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIGS. 1A to 1D illustrate photomicrographs which show that C. albicans CST20 gene complements defects in pseudohyphal growth of ste20/ste20 S. cerevisiae diploid cells.

[0022]FIGS. 2A to 2C show the morphology of S. cerevisiae MATα cells (strain YEL306-1A) deleted for STE20 and CLA4, and transformed with plasmids expressing CLA4 (FIG. 2A), STE20 (FIG. 2B) and C. albicans CST20 (FIG. 2C).

[0023]FIG. 3 shows the nucleotide (SEQ ID NO:5) and predicted amino acid sequences of CST20 (SEQ ID NO:6).

[0024]FIG. 4A is the deletion of CST20 in C. albicans.

[0025]FIG. 4B is the Southern blot analysis with a CST20 fragment from EcoRI to XbaI as a probe.

[0026]FIGS. 5A to 5D show colonies of C. albicans cells grown for 5 days at 37° C. on solid “spider” medium containing mannitol. Wild type strain SC5314 (A), ura3/ura3 cst20Δ/cst20Δ::URA3 strain CDH22 (B), ura3/ura3 cst20Δ/cst20Δ::CST20::URA3 strain CDH36 (obtained by reintegration of CST20 into strain CDH25 by homologous recombination using linearized plasmid pDH190) (C), ura3/ura3 cst20Δ/cst20Δ strain CDH25 transformed with plasmids pYPB1-ADHpt (D). Photomicrographs of representative colonies were taken with a 2× lens (bar=2 mm).

[0027]FIGS. 6A to 6C illustrate virulence assays. Survival curves of mice (n=10 for each C. albicans strain at each inoculation dose) infected with 1×10⁶ (A) and 1×10⁵ (B) cells of C. albicans strains SC5314 (wild type), CAI4 (ura3/ura3), CDH22 (ura3/ura3 cst20Δ/cst20Δ::URA3) (C) Staining of mouse kidney sections with periodic acid Schiff's stain 48 hours after infection with cst20Δ/cst20Δ::URA3 mutant strain CDH22 (a). Some hyphal cells are indicated with arrows (bar=0.1 mm).

[0028]FIG. 7 illustrates the nucleotide (SEQ ID NO:7) and predicted amino acid (SEQ ID NO:8) sequences of CaCLA4.

[0029]FIG. 8A illustrates the deletion of CaCLA4 in C. albicans.

[0030]FIG. 8B illustrates the Southern blot analysis with the CaCLA4 fragment from PstI to XbaI as a probe.

[0031]FIG. 8C illustrates the Northern blot analysis with the CaCLA4 fragment as a probe. PCR with the divergent oligodeoxynucleotides OEL109 and OEL110 was used to delete the coding sequence of CaCLA4. A hisG-URA3-hisG cassette was then inserted, and homologous recombination was used in a two-step procedure to replace both CaCLA4 alleles.

[0032]FIG. 9 illustrates virulence assays. Survival curves of mice (n=15 for each C. albicans strain) infected with 1×10⁶ cells of C. albicans strains SC5314 (wild-type), CDH77 (CaCLA4/cacla4Δ), CLJ1 (cacla4Δ/cacla4Δ) and CLJ5 (CaCla4Δ/cacla4Δ) transformed with the control plasmid pVEC and plasmid pVEC-CaCLA4 carrying the CaCLA4 gene.

[0033]FIG. 10 illustrates the staining of mouse kidney sections with periodic acid Schiff's stain 48 h after infection with C. albicans strains SC5314 and CLJ1.

[0034]FIG. 11 illustrates the nucleotide (SEQ ID NO:9) and predicted amino acid (SEQ ID NO:10) sequences of CaCdc42p.

[0035]FIG. 12 illustrates the nucleotide (SEQ ID NO:11) and predicted amino acid (SEQ ID NO:12) sequences of CaBem1p.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The isolation of proteins that play a role in the viability and/virulence of Candida albicans allows for the development of an assay useful to screen for potential inhibitors of Candida albicans. Each one of the C. albicans' proteins, CaCla4p, Cst20p, Cdc42p and Bem1p, has been found to play such a role. Accordingly, the determination of compounds capable of inhibiting one or more of these proteins would be beneficial in the development of potential anti-fungal agents.

[0037] The CST20 gene of Candida albicans was cloned by functional complementation of a deletion of the STE20 gene in Saccharomyces cerevisiae. CST20 encodes a homolog of the Ste20p/p65^(PAK) family of protein kinases. Colonies of C. albicans cells deleted for CST20 revealed defects in the lateral formation of mycelia on synthetic solid “Spider” media. However, hyphal development was not impaired in some other media. Cells deleted for CST20 were less virulent in a mouse model for systemic candidiasis. Our results suggest that more than one signaling pathway can trigger hyphal development in C. albicans, one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelial formation in asexual C. albicans.

[0038] The CaCLA4 gene of C. albicans was cloned by functional complementation of the growth defect of S. cerevisiae cells deleted for the STE20 and CLA4 genes. CaCLA4 encodes a homolog of the Ste20p family of serine/threonine protein kinases with pleckstrin homology and Cdc42p binding domains in the amino-terminal noncatalytic region. Deletion of both alleles of CaCLA4 in C. albicans caused defects in hyphal formation in vitro in synthetic liquid and solid media, and in vivo in a mouse model for systemic candidiasis. The deletions reduced the invasion of C. albicans cells into kidneys after infection into mice and completely suppressed virulence in the mouse model. Thus, hyphal formation of C. albicans mediated by the CaCla4p protein kinase may contribute to the pathogenicity of this dimorphic fungus.

[0039] The CaBEM1 and CaCDC42 genes of C. albicans were cloned by functional complementation of the growth defect of S. cerevisiae cells deleted for the BEM1 and CDC42 genes, respectively. CaBEM1 encodes an SH3 domain protein with homology to Bem1p, and CaCDC42 encodes a small G-protein with homology to members of the Rho-family of G-proteins.

[0040] With isolation of genes encoding the present C. albicans proteins, namely CaCla4p, Cst20p, Cdc42p, and Bem1p, purified proteins can be made for use in inhibitor assays in accordance with the present invention. Examples of assays utilizing these proteins to identify compounds that inhibit the inherent protein/target interactions of these proteins are described in detail in the specific examples provided herein. As will be appreciated, the protein/target interactions in which these proteins are involved include interactions with each other as well as interactions with independent target compounds.

[0041] Materials and Methods

[0042] Yeast Manipulations

[0043] The yeast form of C. albicans was cultured at 30° C. in YPD medium. Hyphal growth was induced at 37° C. on solid “Spider” media (Liu, H. et al. (1994) Science 266, 1723-1726) containing 1% (w/v) nutrient broth, 0.2% (w/v) K₂HPO₄, 2% (w/v) agar and 1% (w/v) of the indicated sugars (pH 7.2 after autoclaving). Cells were grown in liquid “Spider” media at 30° C. to stationary phase, and then incubated for 5 days at 37° C. on solid “Spider” media at a density of about 200 cells per 80 mm plates. All media were supplemented with uridine (25 μg/ml) for the growth of Ura⁻ strains. Germ tube formation was induced at 37° C. in either 10% fetal bovine serum (GIBCO/BRL) on liquid “Spider” media containing the indicated sugars at an inoculation density of 10⁷ cells per ml.

[0044] Yeast manipulations were performed according to standard procedures.

[0045] Isolation of CST20

[0046] The CST20 gene was isolated from a genomic C. albicans library constructed in plasmid YEp352 from genomic DNA of the clinical isolate WO1 (Boone, C. et al. (1991) J. Bacteriol. 173, 6859-6864). A plasmid carrying an amino-terminally truncated version of CST20 missing the first 918 nucleotides of coding sequence was isolated by screening for suppressors of defects in basal FUS1::HIS3 expression and mating in S. cerevisiae strain YEL64 which was disrupted in STE20. A fragment from nucleotides 958 to 1,252 of CST20 was amplified by the polymerase chain reaction (PCR) and used as a probe to isolate a full length clone by colony hybridization to the C. albicans genomic library transformed into E. coli strain MC1061. Both DNA strands were sequenced by the dideoxy chain termination method. The full-length clone was subcloned between the SacI and HindIII sites of the S. cerevisiae centromere plasmid pRS316 to yield plasmid pRL53.

[0047] Isolation of CaCLA4

[0048] A C. albicans homolog of the S. cerevisiae CLA4 gene was cloned by functional complementation of the growth defect of S. cerevisiae cells that were deleted for the STE20 and CLA4 genes.

[0049] The S. cerevisiae MATα strain YEL257-1A-2 deleted for STE20 and CLA4 and carrying plasmid pDH129 with CLA4 under control of the GAL1 promoter was transformed with the genomic C. albicans library constructed in the S. cerevisiae vector YEp352 carrying URA3 as selectable marker (Boone, C. et al. (1991) J. Bacteriol. 173, 6859-6864). Transformants were grown on selective medium in 4% galactose and then replica-plated to selective medium containing 2% glucose to select for plasmids that were able to support growth in the absence of Cla4p and Ste20p. By screening 1,600 transformants, we isolated plasmid YEp352-CaCLA4 carrying an insert of 5.6 kb with an open reading frame of 2,913 bp capable of encoding a homolog of Cla4p. Subcloning indicated that this open reading frame was responsible for complementation. Both DNA strands were sequenced by the dideoxy chain termination method.

[0050] The open reading frame of the CaCLA4 gene is capable of encoding a protein of 971 amino acids with a predicted molecular weight of 107 kDa and a domain structure characteristic of the Ste20p family of protein kinases (FIG. 7). The catalytic domain present in the carboxyl terminal half of the protein has sequence identities of 74, 63 and 64%, respectively, with S. cerevisiae Cla4p, S. cerevisiae Ste20p and an uncharacterized open reading frame present in the S. cerevisiae genome, 65% with the C. albicans Ste20p homolog Cst20p, and 61% with rat p₆₅PAK (FIG. 7). The amino terminal, noncatalytic region contains a sequence from amino acid residues 69 to 180 with similarity to pleckstrin homology (PH) domains and a sequence from amino acid residues 229 to 292 with 63% identity to the Cdc42p binding domain of S. cerevisiae Cla4p that has been shown to bind the small GTPase Cdc42p (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830). The remaining noncatalytic sequences are less conserved.

[0051] Isolation of CaCDC42

[0052] The S. cerevisiae MATα strain DJTD2-16A carrying the cdc42-1^(ts) mutation was transformed with the genomic C. albicans library constructed in the S. cerevisiae vector YEp352 carrying URA3 as selectable marker (Boone, C. et al. (1991) J. Bacteriol. 173, 6859-6864). Transformants were grown on selective medium at room temperature. Colonies were then replica-plated to selective medium and grown at 34° C. By screening 2,000 transformants, we isolated plasmid YEp352-CaCDC42 carrying an open reading frame of 573 bp capable of encoding a homolog of Cdc42p. Both DNA strands were sequenced by the dideoxy chain termination method. Sub-cloning of various restriction endonuclease fragments indicated that the open reading frame was responsible for complementation of the temperature-sensitive growth defect caused by the cdc42-1^(ts) mutation.

[0053] Construction of C. albicans Strains and Plasmids

[0054] To construct a CST20 null mutant, an EcoRI to SacI fragment from nucleotide positions 989 to 4,134 of CST20 was subcloned into the Bluescript KS(+) vector (Stratagene) to yield plasmid pDH119. A plasmid that contained CST20-flanking sequences from nucleotides 989 to 1,674, and 3,423 to 4,134 joined with BamHI sites, was then created by PCR using the divergent oligodeoxynucleotide primers ODH68 (5′-CGGGATCCAGACCAACCACTCGAACTACT-3′ (SEQ ID NO:1) and ODH69 (5′-CGGGATCCGAAGGTGAACCACCATATTTG-3′ (SEQ ID NO:2); newly introduced BamHI sites are underlined) and plasmid pDH119 as a template. The amplified DNA was cleaved with BamHI and ligated with a 4 kb BamHI to BglII fragment of a hisG-URA3-hisG cassette derived from plasmid pCUB-6 (Fonzi, W. A. & Irwin, M. Y. (1993) Genetics 134, 717-728) to yield plasmid pDH183. This plasmid was linearized with XhoI and SacI and transformed into the Ura⁻ C. albicans strain CAI4 (Fonzi, W. A. & Irwin, M. Y. (1993) Genetics 134, 717-728) to partially replace the coding region of one of the chromosomal CST20 alleles with the hisG-URA3-hisG cassette by homologous recombination. Ura⁺ transformants were selected on Ura⁻ medium, and integration of the cassette into the CST20 locus was verified by Southern blot analysis. Spontaneous Ura⁻ derivatives of two of the heterozygous disruptants were selected on medium containing 5-fluoroorotic acid. These clones were screened by Southern blot hybridization to identify those that had lost the URA3 gene by intrachromosomal recombination mediated by the hisG repeats. This procedure was then repeated to delete the remaining functional allele of CST20.

[0055] A similar procedure was employed to delete the CaCLA4 gene. A 4.6 kb XbaI fragment of YEp352-CaCLA4 was subcloned into the pBluescript KS(+) vector (Stratagene) to yield plasmid pDH205. A plasmid that contained CaCLA4 flanking sequences joined with BglII sites was then created by PCR using the divergent oligodeoxynucleotide primers OEL109 (5′-GAAGATCTTGTAATCAATGTTCCCGTGGA-3′ (SEQ ID NO:3) and OEL110 (5′-GAAGATCTCATCGTGATATTAAATCCGAT-3′ (SEQ ID NO:4); newly introduced BglII sites are underlined) and plasmid pDH205 as template. The amplified DNA was cleaved with BglII and ligated with a 4 kb BamHI-BglII fragment of a hisG-URA3-hisG cassette derived from plasmid pCUB-6 (Fonzi, W. A. & Irwin, M. Y. (1993) Genetics 134, 717-728) to yield plasmid pDH210. This plasmid was linearized with PstI and SacI and transformed into the Ura⁻ C. albicans strain CAI4 (Fonzi, W. A. & Irwin, M. Y. (1993) Genetics 134, 717-728) to replace the coding region of one of the chromosomal CaCLA4 alleles with the hisG-URA3-hisG cassette by homologous recombination. Ura⁺ transformants were selected on Ura⁻ medium, and integration of the cassette into the CaCLA4 locus was verified by Southern blot analysis. Spontaneous Ura⁻ derivatives were then selected on medium containing 5-fluoroorotic acid. These clones were screened by Southern blot hybridization to identify those that had lost the URA3 gene by intrachromosomal recombination mediated by the hisG repeats. This procedure was then repeated to delete the remaining functional allele of CaCLA4.

[0056] To reintegrate CST20 into the genome of mutant strains, the C. albicans integration plasmid pDH190 was constructed by subcloning a KpnI to PstI fragment of CST20 into pBS-cURA3 (pBluescript KS(+) into which the C. albicans URA3 gene was cloned between the NotI and XbaI sites of the polylinker). The integration plasmid was then linearized with NsiI and transformed into C. albicans to target integration into the NsiI site of the CST20Δ::hisG fusion gene. Integrations were selected on Ura⁻ medium and confirmed by Southern blot analysis.

[0057] The C. albicans CST20 expression plasmid pDH188 was constructed by subcloning a SacI to PstI fragment of CST20 into plasmid pVEC carrying a C. albicans autonomously replicating sequence and URA3 as selectable marker. The C. albicans plasmid pVEC-CaCLA4 was constructed by subcloning the KpnI to SacI insert of YEp 352-CaCLA4 into plasmid pVEC.

[0058] Methods Associated with CaCDC42

[0059] Site-directed mutagenesis using the Quickchange Site-Directed Mutagenesis kit from Stratagene was used to generate the following single and double point mutants in CaCDC42 in pJA19; G12V (pSU7, using oligonucleotides OSU5 and OSU6), D118A (pSU9, using oligonucleotides OSU7 and OSU8), C188S (pSU13, using oligonucleotides OSU9 and OSU64), G12V C188S (pSU16) and D118A C188S (pSU18). All constructs were verified by sequence analysis. To overexpress and integrate wild type CaCDC42 and the five mutant CaCDC42 versions in C. albicans, a cassette was made containing 3 kb of hisGURA3hisG sequences (providing an excisable C. albicans selectable marker) isolated from pCUB− (Fonzi and Irwin, 1993) and the 1.4 kb PCK1 promoter (a C. albicans regulatable promoter) isolated from pCA01 (Leuker et al., 1997) yielding pJA24. PCK1 encodes PEP carboxykinase, an enzyme strongly repressed in glucose-containing media (Leuker et al., 1997). PCK1 expression can be induced in media containing a gluconeogenic carbon source such as 2% casamino acids (CAA) media (Leuker et al., 1997). This cassette was cloned into the unique BamHI site in pJA19 and pSU7, 9, 13, 16, 18 generating pJA28 and pSU48, 50, 51, 52, and 45, respectively. Each plasmid was linearized using a unique HpaI site within the PCK1 promoter and was transformed into strains CAI4 (ura3/ura3—Fonzi and Irwin, 1993) or CaDH85 (ura3/ura3CaCDC42/cacdc42::hisG) by either the LiOAc procedure (Schiestl et al., 1993) or the 1-step transformation protocol (Chen et al., 1992). In addition, pJA28 and pSU48 were integrated into a doubly deleted CST20 and CaCLA4 strains CaDH25 and CaLJ5 respectively to create CaSU112 (CST20 double deletion with PCK1CDC42) and CaSU116 (CST20 double deletion with PCK1CDC42^(G12V)) as well as CaSU138 (CaCLA4 double deletion with PCK1CDC42) and CaSU142 (CaCLA4 double deletion with PCK1CDC42^(G12V)). Correct integration was verified by isolating genomic DNA and using PCR with primers OSU43 and OSU45. Integration of PCK1CaCDC42 derivatives was further verified by Southern analysis using the DIG kit from Boehringer Mannheim.

[0060] Construction and Analysis of a CaCDC42 Double Deletion in a PCK1-CDC42 Containing Strain

[0061] A deletion cassette was constructed by removing 640 bp between the KpnI and XbaI sites in pDH208 and replacing this with a BamHI site using oligonucleotides OEL112 plus OEL113, and inserting the hisGURA3hisG cassette to yield pDH212. C.albicans strains CaDH85 and CaSU92 (PCK1CaCDC42) were transformed by either LiOAc procedure or by 1-step protocol using the 9.5 kb SacI-HindIII deletion fragment from pDH212 that contains 478 bp CaCDC42 upstream sequences and coding region, the URA blaster cassette, and 1.8 kb of downstream sequences. Deletion of the second CaCDC42 allele was screened by PCR (three independent products from OSU44 plus OSU8, OSU33 plus OSU34, and ODH103 plus OSU44) and confirmed by Southern analysis; three independent knockouts were found in the first 30 transformants screened in CaSU92 (PCK1CaCDC42) yielding CaSU96-98. No double knockouts were obtained in the 150 CaDH85 transformants screened.

[0062] Phenotypic Analysis

[0063] To assess the effect of Cdc42p depletion on vegetatively grown C. albicans cells, all three of strains CaSU96-98 were grown overnight in 2% CAA (casamino acids) YNB (yeast nitrogen base, Difco)-ura media at 30° C. After 18 hrs of growth the cells were subcultured in either 2% glucose-YNB-ura or 2% CAA YNB-ura and aliquots were stained with DAPI and examined microscopically at various timepoints for up to 10 hrs. To assess the effect of CaCdc42p depletion on hyphal induction, cells were grown for 18 hrs in 2% CAA-ura and were then subcultured under the following conditions: 2% CAA-ura+10% serum at 37° C.; 2% glucose+10% serum at 37° C., and 2% glucose for 4 hrs at 30° C., followed by the addition of 10% serum and incubation at 37° C. Aliquots of each culture were stained with DAPI and examined microscopically every 2-3 hrs.

[0064] FACS Analysis

[0065] Cells were grown in the specified medium and fixed with 70% ethanol for 20 minutes. They were then resuspended in 1 ml of PBS containing 10 μg/ml propidium iodide (Molecular Probes, Eugene, Oreg., USA). The analysis was performed on an EPICS® XL-MCL flow cytometer (Beckman-Coulter) using a 488 nm dichroic filter for side scattering detection and a 620 nm band pass for propidium iodide fluorescence detection. Forward scattering (FS) and side scattering (SS) data were expressed as mean relative units and propidium iodide as mean fluorescence unit, both on a linear scale. Approximately 30,000 cells were analyzed per experiment to monitor the size of CaSU96 and CaSU97 cells either 6 hours or 18 hours after subculture into 2% glucose-ura or 2% CAA-ura.

[0066] PCK1 CaCDC42 Point Mutant Overexpression

[0067] A series of point mutants in CaCDC42 were made in order to assess their function in C. albicans morphogenesis. The point mutations chosen are well defined and the effects have been investigated in other systems (Johnson, 1999). These included the G12V, the D118A and the C188S changes, as well as the G12V-C188S and D118A-C188S double mutants. To assess the effect of the overexpression of PCK1CaCDC42 wild type and mutants, each strain was grown overnight in liquid YNB 2% glucose-ura or YNB 2% CAA-ura. For solid plating the cells were grown in liquid YNB 2% glucose-ura overnight then washed and diluted 10⁻⁵ and 20 μl aliquots were plated onto YNB 2% glucose-ura or YNB 2% CAA-ura and incubated at 30° C. or patched directly on YNB 2% gluc-ura or YNB 2% CAA-ura and incubated at 23° C. For liquid hyphal induction, cells were grown overnight at 30° C. in either YNB 2% glucose-ura or YNB 2% CAA-ura, then subcultured in the same media with the addition of 10% FBS and transferred to 37° C. Hyphal inductions were monitored for up to 6 hrs.

[0068] Cell Staining (DAPI and Calcofluor) and Microscopy Nomarski/Fluorescence)

[0069] Cells were first fixed in 70% ethanol for 20 minutes and rinsed two times in 10 mM phosphate buffer pH 7.4, 150 mM NaCl (PBS) and resuspended in 500 μl PBS before addition of a 1:1000 dilution of either 1 mg/ml 4′,6′-diamino-2-phenylindole (DAPI) or 1 mg/ml Calcofluor white. Cells were stained with either DAPI for 12 minutes or Calcofluor for 5 minutes, then rinsed two times in PBS and examined on a Zeiss Axiophot microscope with a 40× objective under Nomarski optics or under fluorescence conditions, and photographed.

[0070] Molecular Cloning of CaBEM1

[0071] A C. albicans homolog of the CaBEM1 gene was cloned by functional complementation of the growth defect of S. cerevisiae cells deleted for the BEM1 gene. This defect was fully complemented by plasmid YEp352-CaBEM1 carrying the CaBEM1 gene. The open reading frame of the CaBEM1 gene is capable of encoding a protein of 635 amino acids with a domain structure characteristic of Bem1p (FIG. 12). CaBem1p contains two conserved SH3 domains, which are most homologous to the SH3 domains of Bem1p, and also has homology to Bem1p outside of the SH3 domains.

[0072] The S. cerevisiae MATα strain YEL220-1A deleted for BEM1 and carrying plasmid pGAL-BEM1 with BEM1 under control of the GAL1 promoter was transformed with the genomic C. albicans library constructed in the S. cerevisiae vector YEp352 carrying URA3 as selectable marker (Boone, C. et al. (1991) J. Bacteriol. 173, 6859-6864). Transformants were grown on selective medium in 4% galactose and then replica-plated to selective medium containing 2% glucose to select for plasmids that were capable of supporting growth of Bem1p-depleted cells. We isolated plasmid YEp352-CaBEM1 carrying an open reading frame of 1,905 bp fulfilling this criterion and capable of encoding a homolog of Bem1p. Both DNA strands were sequenced by the dideoxy chain termination method, and subcloning of various restriction endonuclease fragments indicated that this open reading frame was responsible for complementation.

[0073] Northern Blot Analyses

[0074] Northern blots of total and poly (A)⁺ RNA from C. albicans cells were performed as described (Leberer, E. et al. (1992) EMBO J. 11, 4815-4824). Signals were quantified by 2-D radioimaging.

[0075] Animal Experiments

[0076] Eight week-old, male CFW-1 mice (Halan-Winkelmann, Paderborn, Germany) were inoculated with 1×10⁵ or 1×10⁶ cells by intravenous injection. Survival curves were calculated according to the Kaplan-Meier method using the PRISM™ program (GraphPad Software Inc., San Diego) and compared using the log-rank test. A P value <0.05 was considered significant.

[0077] To quantify colony-forming C. albicans units in kidneys, mice were sacrificed by cervical dislocation 48 hours after injection and kidneys were homogenized in 5 ml phosphate buffered saline, serially diluted and plated on YNG medium (0.67% yeast nitrogen base, 1% glucose, pH 7.0). Histological examination of kidney sections was done with periodic acid Schiff's stain.

[0078] Results

[0079] Isolation and Characterization of CST20

[0080] A C. albicans homolog of the S. cerevisiae STE20 gene was cloned by functional complementation of the pheromone signaling defect of S. cerevisiae cells that were deleted for the STE20 gene. The mating defect of the STE20 deleted S. cerevisiae strain YEL20 was fully complemented by introduction of the centromeric plasmid pRL53 carrying full length CST20 (mating efficiency was 81±9% in cells expressing CST20, compared with 85±8% in cells expressing STE20; n=3). Similarly, defects in growth arrest and morphological changes in response to pheromone were completely cured by transformation with the CST20 plasmid.

[0081] As shown in FIG. 1, nitrogen deficiency-induced pseudohyphae formation, which is blocked by disruption of STE20 in diploid cells (Liu, H., Styles, C. & Fink, G. R. (1993) Science 262, 1741-1744), was restored by introduction of the CST20 plasmid. Colonies of the diploid STE20 wild type strain L5266 (4) (FIG. 1A) and the isogenic ste20/ste20 strain HLY492 (4) transformed with either the control plasmid pRS316 (FIG. 1B), the CST20 plasmid pRL53 (FIG. 1C), or the STE20 plasmid pSTE20-5 (9) (FIG. 1D) were grown on nitrogen starvation medium (2) for 5 days at 30° C. Photomicrographs were taken with a 4× objective (bar=1 mm).

[0082] As illustrated in FIG. 2, the cytokinesis defect caused by deletion of CLA4, encoding an S. cerevisiae isoform of Ste20p (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830), was not complemented by CST20 (FIG. 2). However, the lethality caused by deletion of both STE20 and CLA4 (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830), could be rescued by CST20 (FIG. 2). The diploid strain YEL306 heterozygous for ste20Δ::TRP1/STE20 cla4Δ::LEU2/CLA4 was transformed with plasmid pRS316 carrying either no insert, CLA4 (pRL21), CST20 (pRL53) or STE20 (pSTE20-5), and then sporulated and dissected. No viable haploid ste20Δ cla4Δ spores were obtained from transformants with the plasmid without insert, but were obtained from transformants with plasmids carrying CLA4 (FIG. 2A), STE20 (FIG. 2B) or CST20 (FIG. 2C).

[0083] Cells were grown to mid-exponential phase in YPD medium at 30° C. No viable ste20Δ cla4Δ segregants were obtained in medium containing 5-fluoro-orotic acid suggesting that the plasmids were essential for viability. Neither STE20 nor CST20 were able to suppress the morphological defect of cla4Δ cells. Photomicrographs were taken by phase contrast with a 40× objective (bar=30 μm).

[0084] The open reading frame of CST20 is capable of encoding a protein of 1,229 amino acids with a predicted molecular weight of 133 kDa and a domain structure characteristic of the Ste20p/p65^(PAK) family of protein kinases (FIG. 3). Numerals at the left margin indicate nucleotide and amino acid positions (FIG. 3). Nucleotide 1 corresponds to the first nucleotide of the initiation codon and amino acid 1 to the first residue of the deduced protein. The putative p21 binding domain has been shadowed, and the kinase domain has been boxed.

[0085] The catalytic domain present in the carboxyl terminal half of the protein has sequence identities of 76 and 56%, respectively, with S. cerevisiae Ste20p (Leberer, E. et al. (1992) EMBO J. 11, 4815-4824) and Cla4p (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830). The amino terminal, non-catalytic region contains a sequence from amino acid residues 473 to 531 with 68% identity to the p21 binding domain of Ste20p that has been shown to bind the small GTPase Cdc42p. This region contains the sequence motif ISxPxxxxHxxH thought to be important for the interaction of the p21 binding domain with the GTP-bound forms of Cdc42Hs and Rac1 (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830). The remaining non-catalytic sequences are less conserved. Unique sequences not present in Ste20p and the other members of the family are found at the amino terminus and between the p21 binding and catalytic domains.

[0086] A CST20 transcript of 4.9 kb in size was detected in Northern blots. This transcript was present at similar levels in yeast cells grown in YPD at room temperature and germ tubes induced by a temperature shift to 37° C. Chromosomal Deletion of CST20

[0087] Homologous recombination was used in a multistep procedure to partially delete CST20 in a URA⁻ C. albicans strain (FIG. 4A). PCR with the divergent oligodeoxynucleotides ODH68 and ODH69 was used to partially delete the coding sequence of CST20. AhisG-URA3-hisG cassette was then inserted. The deletion was confirmed by Southern blot analyses (FIG. 4B). The genomic DNA samples digested with XhoI were from following strains: Lane #1, CAI4 (ura3/ura3 CST20/CST20); lane 2, CDH15 (ura3/ura3 CST20/cst20Δ::hisG-URA3-hisG); lane 3, CDH18 (ura3/ura3 CST20/cst20Δ::hisG); lane 4, CDH22 (ura3/ura3 cst20Δ::hisG-URA3-hisG/cst20Δ::hisG); lane 5, CDH25 (ura3/ura3 cst20Δ::hisG/cst20Δ::hisG). Northern blots showed that the CST20 transcript was absent in the corresponding homozygous deletion strains.

[0088] The lateral outgrowth of hyphae from colonies grown on solid “Spider” media containing mannitol or sorbitol was completely blocked by deletion of CST20 (FIG. 5B).

[0089] Mycelial formation was drastically reduced when the media contained galactose, mannose or raffinose. The mutant strains regained the ability to form hyphae when wild type CST20 was reintroduced by transformation with the CST20 expression plasmid pDH188 or reintegrated into the genome by targeted homologous recombination (FIG. 5C). The CST20 transcript was detected in these strains by Northern blot analysis.

[0090] Mutant strains formed hyphae when colonies were grown on “Spider” media containing either glucose or N-acetyl glucosamine. Normal hyphae formation was also observed on rice agar and on agar containing Lee's medium or 10% serum. The frequency of germ-tube formation in either liquid Lee's medium, 10% serum or liquid “Spider” media containing any of the sugars tested above, were also normal. These results indicate that Cst20p is not required for hyphae formation under all conditions but are involved in the lateral formation of mycelia on some solid surfaces.

[0091] Chromosomal Deletion of CaCLA4

[0092] Homologous recombination was used in a multistep procedure to delete both alleles of CaCLA4 in C. albicans (FIG. 8A). FIG. 8A shows the restriction endonuclease map of CaCLA4. The coding sequence is indicated by the arrow. PCR with the divergent oligodeoxynucleotides OEL109 and OEL110 was used to delete the coding sequence of CaCLA4. A hisG-URA3-hisG cassette was then inserted and a two-step procedure was used to delete both alleles of CaCLA4 by homologous recombination. The endonuclease restriction sites are as follows: B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; P, PstI; S, SacI; X, XbaI. The deletions were confirmed by Southern blot analyses (FIG. 8B). Southern blot analysis with a 1.1 kb CaCLA4 fragment from PstI-XbaI as a probe. The genomic DNA samples digested with EcoRI were from following strains: Lanes: 1, CAI4 (ura3/ura3 CaCLA4/CaCLA4); 2, CDH77 (ura3/ura3 CaCLA4/cacla4Δ::hisG-URA3-hisG); 3, CDH88 (ura3/ura3 CaCLA4/cacla4Δ::hisG); 4, CLJ1 (ura3/ura3 cacla4Δ::hisG-URA3-hisG/cacla4Δ::hisG); and 5, CLJ5 (ura3/ura3 cacla4Δ::hisG/cacla4Δ::hisG). Northern blots showed that the CaCLA4 transcript with a size of 4.1 kb was reduced to about 40% in heterozygous CaCLA4/cacla4Δ cells and was absent in homozygous cacla4Δ/cacla4Δ deletion cells (FIG. 8C). The transcript was present at about wild-type levels when the CaCLA4 gene was retransformed into the homozygous deletion cells by using an autonomously replicating plasmid carrying the CaCLA4 gene (FIG. 8C). Northern blot analysis of poly(A)⁺ RNA isolated from following strains grown in the yeast form in YPD at 30° C.: Lanes: 1, SC5314 (wild-type); 2, CDH88; 3, CLJ5 transformed with pVEC; 4, CLJ5 transformed with pVEC-CaCLA4. The blot was probed with fragments specific for CaCLA4 (upper panel) or CaACT1 (lower panel) and quantified by radioimaging. Numbers at the bottom of the figure depict the relative amounts of CaCLA4 transcript in relation to the amounts of CaACT1 transcript (mean values of two independent experiments).

[0093] It was found that viability of C. albicans cells was not affected by deleting either one or both alleles of CaCLA4. Mutant cells showed the same growth behavior as wild-type cells, independently whether the cells were grown under conditions favoring either the yeast or filamentous forms. However, deletion of both CaCLA4 alleles generated defects in cellular morphology producing a heterogeneous population of aberrantly shaped cells that were frequently multibudded and multinucleated. This phenotype indicates a defect in cytokinesis resembling the phenotype of S. cerevisiae cells deleted for CLA4 (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830).

[0094] Deletion of both CaCLA4 alleles caused defects in hyphal formation in all media and under all conditions that we investigated. When morphological switching was induced in liquid media by either serum, N-acetyl glucosamine, proline, pH increase, temperature shift, or Lee's medium, wild-type cells and cells deleted for only one or both alleles of CaCLA4 produced germ tubes after about 30 minutes. In wild-type cells and cells deleted for only one allele of CaCLA4, these germ tubes elongated and grew into long hyphae after prolonged incubation. Cells deleted for both alleles of CaCLA4 failed to produce hyphae, however. Instead, these cells produced multiple short protrusions giving rise to an aberrant morphology.

[0095] On solid media containing either serum, rice agar or mannitol, the normal formation of mycelia was completely suppressed by deletion of both CaCLA4 alleles. This phenotype was reversed by introducing the CaCLA4 gene on a plasmid, and deletion of only one allele had no effect.

[0096] Virulence Studies

[0097] To determine the role of Cst20p for virulence, mice were injected intravenously with wild type and mutant strains and monitored for survival and for fungal invasion into kidneys. We found that the Ura⁻ strain CAI4 was not pathogenic (FIGS. 6A and B). However, infection with Ura⁺ wild type cells resulted in rapid mortality with a rate that was dependent on the dose of injected cells (1×10⁶ cells in FIG. 6A, and 1×10⁵ cells in FIG. 6B). Survival was significantly prolonged, however, in mice infected with Ura⁺ cells deleted for both alleles of CST20 (cst20Δ/cst20Δ::URA3). This effect, which was reproducible and statistically significant, was observed at high (FIG. 6A) or low (FIG. 6B) doses of infection (with P values of 0.027 and 0.001, respectively) and correlated with colony-forming units per kidney (1.5×10⁶ for wild type cells and 7×10⁵ for cst20Δ/cst20Δ::URA3 mutant cells) after 48 hours of infection with 1×10⁶ cells. These effects on virulence could be reversed by reintroducing CST20 into the strain deleted for both CST20 alleles, and were not observed in Ura⁺ cells deleted for only one CST20 allele. A histological examination revealed that cells deleted for both alleles of CST20, were able to form hyphae in infected kidneys (FIG. 6C).

[0098] To investigate whether CaCla4p is required for virulence, mice were injected intravenously with wild-type and mutant C. albicans strains and monitored for survival and for fungal invasion into kidneys. Infections with CaCLA4 wild-type cells (strain SC5314) resulted in rapid mortality (FIG. 9). No difference in the mortality rate was observed after infection with cells deleted for only one allele of CaCLA4 (strain CDH77). All mice survived, however, after infection with cells deleted for both alleles of CaCLA4 (strain CLJ1 and CLJ5pVEC1). This effect correlated with a reduction in the amount of colony-forming units per kidney of infected animals and was reversed by transformation of the cells with a plasmid carrying the CaCLA4 gene (strain CLJ5CaCLA4) (FIG. 9). A histological examination revealed that kidneys from mice injected with either wild-type cells or cells deleted for one allele of CaCLA4 were heavily infected with C. albicans cells that produced hyphae densely penetrating the animal tissue (FIG. 10, left panel), whereas kidneys from mice injected with cells deleted for both CaCLA4 alleles contained small foci of aberrantly shaped cells that frequently carried multiple protrusions (FIG. 10, right panel). The morphologies of these cells were similar to those induced by serum under in vitro conditions. Thus, the function of CaCla4p is required for morphological switching of C. albicans under in vitro and in vivo conditions and for virulence.

[0099] Results Associated with CaCDC42

[0100] Molecular Cloning of the CaCDC42 Gene

[0101] A C. albicans homolog of the CaCDC42 gene was cloned by functional complementation of the temperature-sensitive growth defect of S. cerevisiae cells carrying the cdc42-1^(ts) mutation. The growth defect was fully complemented by plasmid YEp352-CaCDC42. The open reading frame of the CaCDC42 gene is capable of encoding a protein of 191 amino acids with homology to the Rho-family of small G-proteins (FIG. 11). The highest homology is found with Cdc42p from S. cerevisiae.

[0102] CaCdc42p is Required for Vegetative Growth.

[0103] To investigate CaCDC42 function in the yeast form of C. albicans, both copies of CaCDC42 were deleted in a strain containing a copy of CaCDC42 under the control of the PCK1 promoter. This strain was subcultured in liquid media that represses PCK1 expression (2% glucose-ura) and monitored microscopically at various timepoints after subculture. By 6 hrs the majority of the culture had arrested as large, round, unbudded cells suggesting that CaCdc42p is necessary for bud formation and for polarized growth. By fluorescence activated cell sorting (FACS) analysis, the average size of CaSU96 or CASU97 after 6 hours in 2% gluc-ura was increased by 2.3 fold and 2.1 fold respectively. 4′,6′-diamino-2-phenylindole (DAPI) staining was used to determine the nuclear content of the CaCdc42p depleted cells. The majority of the arrested cells contained two nuclei, and the proportion of binucleate cells remained essentially constant upon continued inhibition of CaCdc42p function. Monitoring the cultures by FACS analysis showed the size of CaSU96 or CASU97 cells continued to increase to 3.4 fold and 4.7 fold respectively in glucose media compared to cells grown in casamino acids (CAA) after 18 hours. In addition, cellular polarity was investigated by staining cells with calcofluor to monitor chitin distribution. In contrast to wild type strains where chitin staining was primarily localized to bud sites, the CaCdc42p depleted cells were round and showed delocalized calcofluor staining.

[0104] Overexpression of Cdc42 Point Mutants in Vegetatively Grown Cells

[0105] As described in Materials and Methods, wild type CDC42 and a series of point mutant derivatives were fused to the PCK1 promoter and integrated into either strain CAI4, which contains both wild type copies of CaCDC42, or into strain CaDH85, which contains one copy of CaCDC42 disrupted. The G12V mutation has decreased intrinsic GTPase activity resulting in a mutant protein locked in an activated GTP-bound state (Ziman et al., 1991), while the D118A mutant protein does not undergo GDP to GTP exchange resulting in a protein locked in the inactive GDP-bound (Ziman et al., 1991). The C188S mutant protein cannot be isoprenylated and therefore is not localized to the membrane (Ziman et al., 1991). These cells were grown overnight in PCK1 inducing media (2% CAA-ura), stained with DAPI and examined microscopically. Overexpression of the wild type CaCdc42p in strain CaDH85 had no effect on cell proliferation or cell morphology; cells both formed colonies after overnight growth on solid medium and looked normal. In contrast, overexpression of either the hyperactive CaCdc42^(G12V) protein or the dominant negative D118A protein in strain CaDH85 blocked cell proliferation. The PCK1 mediated expression of the G12V allele generated aberrant multibudded cells while overexpression of the dominant negative D118A protein resulted in large, round cells that accumulated nuclei. The proliferation arrest and the cell morphology changes caused by overexpression of the hyperactive and dominant negative alleles require a functional CaCdc42p CAAX box. The C188S mutation rescued the unviability caused by overexpression of the G12V and D118A allele proteins, and generated morphologically normal cells (data not shown). In strain CAI4 that contains the two wild type copies of CaCDC42 in addition to the PCK1 driven gene, the phenotypic effects of the mutant proteins are similar but not as severe.

[0106] Overexpression of CaCdc42p^(G12V) in a CST20-Deleted Strain or CaCLA4-Deleted Strain

[0107] PCK1CaCDC42 (pJA28) and PCK1CaCDC42^(G12V) (pSU48) were integrated into strains doubly deleted for either CST20 (Leberer et al., 1996) or CaCLA4 (Leberer et al., 1997b) and the phenotypes were examined under PCK1 inducing or non-inducing conditions. Overexpression of CaCdc42p^(G12V) in a CaCLA4 deficient strain resulted in unviable cells that were severely elongated and multibudded; prolonged incubation lead to extensive lysis of the cells. This was not observed in the CaSTE20-deleted strain overexpressing CaCdc42p^(G12V). A minor growth defect was observed on solid media but the cells appeared normal upon microscopic analysis. There was no significant effect of overexpressing CaCDC42 in either of these strains. The influence of the PCK1CaCDC42^(G12V) allele on cell growth was temperature dependent; room temperature expression had a more detrimental effect on proliferation than expression at 30° C., although at either temperature the CST20 deleted strain grew better than either the control or the CaCLA4 deleted strain.

[0108] CaCdc42p Involvement in Hyphal Formation

[0109] A time course experiment was done to examine the effect of depletion of CaCdc42p on hyphal formation (as described in Materials and Methods). After CaCdc42p is largely depleted (incubation for 4 hrs in glucose-ura and then hyphal formation is induced with 10% FBS for 2 hrs), the cells arrest and are large, round, and primarily binucleate. If the cells are immediately switched to 2% glucose-ura+10% serum, short germ tubes are initiated but then stop as CaCdc42p is depleted. These cells also increase in size and arrest with primarily two nuclei.

[0110] Discussion

[0111] In S. cerevisiae, Ste20p fulfills multiple functions during mating (Leberer, E. et al. (1992) EMBO J. 11, 4815-4824), pseudohyphae formation (Liu, H., Styles, C. & Fink, G. R. (1993) Science 262, 1741-1744), invasive growth (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985) and cytokinesis (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830). CST20 expression in S. cerevisiae fully complements these functions. Thus, Cst20p has the potential to fulfill similar functions in C. albicans.

[0112] The yeast-to-hyphal transition of C. albicans is a morphological change that can be triggered by a wide variety of factors. Carbohydrates, amino acids, salts, and serum have been described as inducers of germ tube formation, as have pH changes, temperature increases and starvation, but no single environmental factor could be defined as uniquely significant in stimulating the morphological switch. Hence C. albicans appears capable of responding to many divergent environmental signals. Disruption of both CPH1 alleles, which encode a homolog of the S. cerevisiae Ste12p transcription factor (Liu, H. et al. (1994) Science 266, 1723-1726), suppressed the lateral formation of mycelia from colonies grown on solid “Spider” medium, but did not block hyphal development in other media. We have shown that C. albicans mutant cells deleted for CST20 display a similar phenotype, and that the effect of these mutations on hyphal development is dependent on the carbon source in which the cells were grown.

[0113] These observations are consistent with the idea that several signaling pathways can trigger morphogenesis in C. albicans. Furthermore, the behavior of C. albicans mutant strains deleted for either CPH1 or CST20 indicates that these pathways might operate independently to activate hyphal development under differing environmental conditions. C. albicans encounters a variety of different microenvironments during the development of superficial and systemic infections. Hence, the existence of parallel morphogenetic signaling pathways might provide a distinct advantage to this pathogen.

[0114] The results indicate that the pathway controlled by Cst20p is not essential for virulence in a mouse model of systemic infections. It is not inconceivable that this pathway plays a role in other forms of infections, for example in the development of superficial infections of the mucosal epithelia (thrush). An as yet undefined role of Cst20p in pathogenicity outside of the Cst20 signaling pathway is suggested, however, by prolonged survival of mice infected with cst20 deleted cells. It is unlikely that this effect is caused by defects in hyphal formation since a histological examination of infected kidneys revealed that the CST20 deleted cells are not restricted in their capacity to form hyphae.

[0115] In S. cerevisiae, Cla4p plays a role in cytokinesis and shares with Ste20p an essential function for polarized growth during budding (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830). Cla4p binds the Rho-like small G-protein Cdc42p (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830), which is involved in controlling cell polarity during budding and in response to pheromone. Like Ste20p and the mammalian homolog p21-activated kinase (p65^(PAK)), Cla4p is able to phosphorylate and activate myosin-I, a mechanism that may contribute to the organization of the actin cytoskeleton.

[0116] The finding that CaCLA4 expression in S. cerevisiae completely complements the Cla4p functions suggests that CaCla4p may have similar properties in C. albicans. Thus, CaCla4p may be required for myosin-I driven polarized growth during hyphal formation in a mechanism that may involve the C. albicans homolog of Cdc42p. Our complementation assays in S. cerevisiae suggest that CaCla4p may share an essential function with Cst20p, the C. albicans homolog of Ste20p (FIGS. 6A and 6B). This notion suggests, together with our findings that null mutants of CaCLA4 are completely non-pathogenic (FIG. 10) and null mutants of CST20 are reduced in virulence (FIGS. 6A and 6B), that CaCla4p and Cst20p, and proteins such as CaCdc42p and CaBem1p interacting with these protein kinases, may be valid targets for the development of antifungal agents.

[0117] In Candida albicans the Homolog of the Cdc42p GTPase, CaCdc42p, is Required for Proper Proliferation and Cellular Morphogenesis

[0118] Construction of a strain with the only functional copy of the CaCDC42 gene under control of the regulated PCK1 promoter permitted the controlled shut-off of CaCDC42 expression. Vegetatively growing cells that were no longer expressing CaCDC42 ceased proliferation within several hours of promoter shut-off. Continued inhibition of CaCDC42 expression resulted in an accumulation of large, round, unbudded cells. These cells showed delocalized chitin staining, in contrast to the wild type cells that localized chitin to the bud necks and bud scars.

[0119] Because of the importance of the Rho family GTPases in the general regulation of polarized morphogenesis in eukaryotic cells (Tanaka and Takai, 1998; Valster et al., 2000; Chimini and Chavrier, 2000), we further investigated the involvement of CaCDC42 in the control of hyphal growth in C. albicans.

[0120] In C. albicans, specific external signals such as high temperature and serum are capable of inducing an essentially quantitative shift of yeast-like cells into hyphal forms (Brown and Gow, 1999; Whiteway, 2000). When C. albicans cells were depleted of CaCdc42p prior to triggering the yeast to hyphal switch, the cells arrested with phenotypes similar to that of the yeast form mutants, namely large, round, unbudded cells. When the yeast to hyphal switch was triggered at the same time as repression of CaCDC42 expression, the cells formed abortive germ tubes but then arrested as primarily binucleate, unbudded cells. This result suggests that the germ tube initiates before the CaCdc42p is depleted, but that in the absence of continuing CaCdc42p function the germ tube cannot be extended. Thus CaCDC42 appears essential for proper polarized growth of C. albicans cells growing under both yeast and hyphal inducing conditions.

[0121] As was also noted in the yeast cell growth situation, the phenotype of the dominant negative D118A allele was not identical to the null in hyphal conditions. The D118A mutant generates aberrant hyphal structures in the presence of serum, while the null mutant blocks hyphal formation under these conditions. It is possible that the growth conditions for stimulating hyphal formation are somewhat incompatible with PCK1 expression, and therefore the expression levels of CaCdc42p^(11A) are insufficient to block endogenous GTPase function when the cells are grown in the presence of serum. Alternatively, it may be that the target of the dominant negative protein plays a different role in yeast and hyphal growing cells.

[0122] The present work shows that CaCdc42p is required for both yeast and hyphal proliferation. In addition, this work establishes that simple overexpression of CaCDC42 has little effect on the cells, while modification of CaCdc42p activity through activating and dominant negative mutations has profound cellular effects. In the case of the hyperactive G12V allele it is evident that a primary effector is the Cst20p kinase. Thus it is likely that the activity of the CaCdc42p GTPase, rather than its expression, is the primary means through which CaCdc42p influences cellular morphogenesis.

[0123] The present invention will be more readily understood by referring to the following examples, which are given to illustrate the invention rather than to limit its scope.

EXAMPLE I Screening Test for Inhibitors of CaCla4p and Cst20p

[0124] An in vitro assay can be used to identify compounds that inhibit the activity of the proteins CaCla4p and/or Cst20p. Inhibitors of these proteins are potentially useful to render pathogenic fungi avirulent.

[0125] Since CaCla4p and/or Cst20p are kinases, a standard kinase assay is suitable to identify compounds that inhibit their activity. In this example, myelin basic protein is used as a substrate. Such an assay is well-known in the art as described in (Wu et al., J. Biol. Chem., 1995). Under uninhibited conditions, the myelin basic protein is phosphorylated in the presence of a kinase (such as Cla4p and Cst20p) and ATP as a source of phosphate. This reaction is monitored by using labeled ATP. In the presence of a compound that inhibits kinase activity, the substrate will not be phosphorylated and this will be detected by an absence of labeled substrate in the reaction mixture.

[0126] Following the identification of an inhibitor of Cla4p and/or Cst20p, the effect of this inhibitor on the endogenous human protein kinase, p65^(PAK), must be determined. This is determined also using a standard kinase assay as described above, substituting p65^(PAK) for Cla4p and/or Cst20p. For use as an anti-fungal agent, the desired inhibitor will selectively inhibit CaCla4p and Cst20p and not the endogenous p65^(PAK) of the patient being treated.

[0127] However, compounds inhibiting all three proteins, namely, CaCla4p, Cst20p and p65^(PAK), would be useful in the treatment of inflammation.

EXAMPLE II Screening Test for Inhibitors of CaCdc42p

[0128] An in vitro assay can be used to identify compounds that inhibit the activity of the protein CaCdc42p. Inhibitors of this protein are also potentially useful to render pathogenic fungi avirulent.

[0129] Since CaCdc42p is a GTPase, a standard GTPase assay is used to identify compounds that inhibit GTPase activity. Accordingly, in uninhibited conditions, CaCdc42p will hydrolyze GTP to form GDP. In the presence of an inhibitor, no hydrolysis of GTP will occur. This is monitored by using labeled GTP. Compounds found to inhibit GTP hydrolysis, and thus, to inhibit CaCdc42p, are potentially useful as anti-fungal agents.

EXAMPLE III Screening Test for Inhibitors of CaBem1p

[0130] An in vitro assay can be used to identify compounds that inhibit the activity of the protein CaBem1p. Inhibitors of this protein are also potentially useful to render pathogenic fungi avirulent.

[0131] CaBem1p binds protein kinases such as CaCla4p and Cst20p. A kinase-binding assay as described in detail in Example IV would be useful to identify compounds that inhibit this interaction.

EXAMPLE IV

[0132] Screening Test for Inhibitors of CaCla4p and CaCdc42p Interaction

[0133] In view of the indicated interaction between protein kinases in C. albicans, such as CaCla4p and Cst20p, and other proteins, such as CaCdc42p and CaBem1p, and the role these interactions are believed to play in the viability and virulence of C. albicans, inhibitors of such interactions are also potentially useful as anti-fungal agents. Accordingly, an in vitro assay to screen for such inhibitory compounds would be useful.

[0134] In this example, a kinase-regulator interaction assay can be used to determine inhibition between the protein kinase, CaCla4p or Cst20p, and proteins that it interacts with (interacting proteins), such as CaCdc42p and CaBem1p. The protein kinase is solid phase bound and the interacting proteins are in suspension free to interact with the protein kinase. A labeled antibody specific to the interacting protein is added to the reaction mixture. In uninhibited conditions, the interaction between interacting protein and protein kinase will occur. The labeled antibody will then bind to the bound interacting protein and this will be detected in the form of bound label. However, in the presence of an inhibiting compound, the interaction will not occur and this will be detected by the absence of bound label.

EXAMPLE V A Two-Hybrid CaCdc42p and CaCla4p Interaction System in a Humanized S. cerevisiae Strain

[0135] This screening assay is based on the assumption that the interaction of the small G-protein CaCdc42p with its cellular targets Cst20p and CaCla4p is essential for viability of C. albicans cells. This assumption is reasonable based on work that has been performed in S. cerevisiae (Leberer E. et al. (1997) Embo J. 16, 83-97). This assay will be useful to detect inhibitors of this interaction and potential anti-fungal agents.

[0136] In this two-hybrid interaction system, the gene for green fluorescent protein is fused to the GAL1 promoter to provide a functional read out. This reporter gene will be integrated into a S. cerevisiae strain in which the STE20 and CLA4 genes have been replaced by the human homolog p65PAK. The CaCDC42 gene will be fused to the DNA binding domain of GAL4, and the CaCLA4 gene will be fused to the activation domain of GAL4. Interaction of the two proteins will cause green fluorescence, whereas inhibitors of the interaction will suppress fluorescence.

[0137] Non-specific inhibitors of the two-hybrid interaction system will be excluded by performing a parallel screen with unrelated fusion proteins known to interact. Compounds of general toxicity or inhibitors of the human homologs will also be excluded in this system because those compounds will not allow growth of the cells and therefore reduce the fluorescent readout in both parallel screens.

[0138] For use in the assay, a two-hybrid yeast strain carrying the GAL4-GFP fusion gene is constructed. This strain will be deleted for the CLA4 gene using the TRP1 marker as described (Leberer E. et al. (1997) Embo J. 16, 83-97). The STE20 gene will be replaced by the human PAK gene as described above. To replace the CDC42 gene by its human homolog, an integrating plasmid will be constructed carrying the HsCDC42 gene fused to a URA3 blaster gene and CDC42 flanking sequences. After linearization, the construct will be transformed into the PAK-containing two-hybrid strain, and integrants will be selected on -ura medium. The URA3 gene will then be looped out on FOA medium. The various gene disruptions and gene replacements will be verified by Southern blot analyses.

[0139] The two-hybrid vectors carrying the CaCDC42 gene fused to the GAL4-DNA binding domain and the CaCLA4 gene fused to the transcriptional activation domain of GAL4 will be constructed by standard procedures. To facilitate the interaction of the two proteins, site-directed mutagenesis is used to create a mutation in the CAAX-box domain of CaCDC42p to prevent isoprenylation and targeting of the fusion protein to the plasma membrane. For use in high-throughput screening, the present assay will be evaluated, optimized and adapted to achieve suitable conditions.

EXAMPLE VI Detection of the Presence of C. albicans Using Probes

[0140] The sequences of either one of the genes CaCLA4, CST20, CaCDC42 and CaBEM1 may be used to derive probes for the detection of C. albicans using PCR techniques or hybridization assays.

EXAMPLE VII Use of Nucleotide Sequences of CaCLA4, CST20, CaCDC42 and CaBEM1 to Identify Homologue from other Fungi

[0141] The nucleotide sequences of CaCLA4, CST20, CaCDC42 and CaBEM1 may be used to identify and clone homologues from other fungi.

EXAMPLE VIII A S. cerevisiae-Based Screening System Using CaSte20p and the Pheromone Signaling Pathway as Drug Target

[0142] In this system, green fluorescent protein (GFP) under transcriptional control of a pheromone inducible promoter (FUS1) is used as a read out. The pheromone signaling pathway and thereby the reporter gene will be induced with pheromone in two different strains: firstly, in a strain in which STE20 is functionally replaced by the CST20 gene, and secondly, in a strain in which STE20 is functionally replaced by the mammalian homolog PAK. Compounds that block the induction of the reporter gene in the CaSTE20 strain but not in the PAK strain are expected to be specific inhibitors of the C. albicans kinase. In fact, CST20 gene is part of a pathway that links pheromone to gene expression. This dual assay represents a means by which compounds selective for CST20 can be identified, while eliminating those compounds which exhibit inhibitory action against the mammalian homolog PAK or compounds of general toxicity.

[0143] To conduct the foregoing assay, the FUS1 gene, including its promoter, is isolated by the polymerase chain reaction (PCR) from genomic DNA of S. cerevisiae and fused to the GFP gene from Aequoria victoria on a yeast expression plasmid. The function of the reporter gene is analyzed after transformation of a MATa yeast strain and induction with pheromone.

[0144] The STE20 gene is replaced in a supersensitive sst1 yeast strain by the human PAK gene using homologous recombination. For this purpose, an integrating plasmid is constructed carrying the PAK gene fused to URA3 blaster and STE20 flanking gene sequences. The construct is linearized and transformed into yeast, and integrants are selected on -ura medium. The URA3 gene is then looped out on FOA medium to gain back the ura3 marker. Correct integration of the PAK gene is confirmed using Southern blot analysis.

[0145] The humanized strain is then transformed with the FUS1-GFP reporter gene and analyzed for a functional signaling pathway by measuring green fluorescence after induction with pheromone. For use identifying inhibitors of C. albicans, this assay system will be evaluated, optimized and adapted to the scale for high-throughput screening.

EXAMPLE IX Fluorescence Resonance Energy Transfer (FRET) as Probe for Protein-Protein Interactions

[0146] The engineering of different GFP mutants with altered fluorescence characteristics allows the use of fluorescence resonance energy transfer (FRET) to probe protein-protein interactions (Heim and Tsien (1996) Curr. Biol. 6, 178-182). The FRET phenomenon consists in a fluorescence transfer between a donor and a receptor fluorochrome. If excitation and emission wavelengths are compatible, the FRET is easily measurable. The main parameter of the reaction is the distance between donor and receptor, which must be in the range of nanometers. This is precisely the kind of values in protein-protein interactions.

[0147] A novel yeast assay system can be developed which uses FRET to measure the in vivo interaction between CaCdc42p and Cacla4p. The CaCDC42 gene will be fused to a GFP mutant that acts as donor, and the CaCLA4 gene will be fused to a mutant that acts as receptor. The yeast strain used as an expression system will be humanized as described in Example VIII. Inhibitors of the interaction are expected to reduce energy transfer, and this reduction can readily be measured spectroscopically. The interaction of unrelated proteins known to interact will be used as a reference to exclude non-specific inhibitors of the assay system. Compounds inhibiting the interaction of the human homologs or of general toxicity will be excluded by inhibition of growth and therefore reduced fluorescence in both screens.

[0148] To conduct such an assay, the CaCDC42 gene will be fused to the gene encoding the GFP^(Y66H) mutant as donor, and the CaCLA4 gene will be fused to the gene encoding the GFP^(S65T) mutant as receptor (Heim and Tsien (1996), Curr. Biol. 6, 178-182). The constructs will then be transformed into the humanized yeast strain described in Example VIII, and the FRET phenomenon will be analyzed in yeast cultures using fluorescence spectroscopy. The conditions for the assay will be worked out and optimized. The assay conditions will be adapted for use in microtiter plates for automated screening.

[0149] While the invention has been described in connection with specific embodiments thereof, it will be understood that further modifications are possible. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

1 12 1 29 DNA Artificial Sequence primer ODH68 1 cgggatccag accaaccact cgaactact 29 2 29 DNA Artificial Sequence primer ODH69 2 cgggatccga aggtgaacca ccatatttg 29 3 29 DNA Artificial Sequence primer OEL109 3 gaagatcttg taatcaatgt tcccgtgga 29 4 29 DNA Artificial Sequence primer OEL110 4 gaagatctca tcgtgatatt aaatccgat 29 5 4492 DNA Candida albicans CDS (355)...(4044) 5 gtacccactt tacaatcact tacaagtcaa ataattacaa cttgacaatc ctcactttaa 60 gtctaacgta tatacgcgta caccatctta tactccacat acatattgga ttcaattttt 120 attttattgt ttagtttata tccaaccact gacaattacc aatagttttc aattaatatt 180 cacaatttaa ctatttgttt gacagctgaa aagagataaa aaaagaatca agtgctataa 240 ctcacaaggg ctagaaataa gtttgcaaaa aacaagtttt aaaaatagta actgcacttt 300 tgttgactct ttcacctccc cattgaattt aactgaacac aaataaagcc tatc atg 357 Met 1 agc ata ctt tca gag aac aat cct aca cca aca tca ata aca gat cca 405 Ser Ile Leu Ser Glu Asn Asn Pro Thr Pro Thr Ser Ile Thr Asp Pro 5 10 15 aat gag tct tct cat cta cac aac cca gag tta aac tct gga acg agg 453 Asn Glu Ser Ser His Leu His Asn Pro Glu Leu Asn Ser Gly Thr Arg 20 25 30 gtt gct tct gga cct gga cct gga cct gaa gtt gaa tca aca cca cta 501 Val Ala Ser Gly Pro Gly Pro Gly Pro Glu Val Glu Ser Thr Pro Leu 35 40 45 gca ccc cca act gag gtc atg aat act aca tca gct aat act tct tca 549 Ala Pro Pro Thr Glu Val Met Asn Thr Thr Ser Ala Asn Thr Ser Ser 50 55 60 65 tta agt tta ggg tct cca atg cac gag aaa ata aaa caa ttt gat caa 597 Leu Ser Leu Gly Ser Pro Met His Glu Lys Ile Lys Gln Phe Asp Gln 70 75 80 gac gag gtt gac act ggg gaa act aat gat agg act ata gaa tct gga 645 Asp Glu Val Asp Thr Gly Glu Thr Asn Asp Arg Thr Ile Glu Ser Gly 85 90 95 tct agt gat att gat gat tca caa caa tca cat aac aac aac aac aac 693 Ser Ser Asp Ile Asp Asp Ser Gln Gln Ser His Asn Asn Asn Asn Asn 100 105 110 aac aac aac aac aac aac gag agc aat cca gaa tca agt gaa ggc gat 741 Asn Asn Asn Asn Asn Asn Glu Ser Asn Pro Glu Ser Ser Glu Gly Asp 115 120 125 gat gaa aaa acc caa gga atg cct cct cga atg cca ggg aca ttc aat 789 Asp Glu Lys Thr Gln Gly Met Pro Pro Arg Met Pro Gly Thr Phe Asn 130 135 140 145 gtg aaa ggt ttg cac caa ggg gat gat agt gac aat gaa aaa cag tac 837 Val Lys Gly Leu His Gln Gly Asp Asp Ser Asp Asn Glu Lys Gln Tyr 150 155 160 acc gag cta act aaa tca atc aat aaa cgt acc agt aaa gat tcg tat 885 Thr Glu Leu Thr Lys Ser Ile Asn Lys Arg Thr Ser Lys Asp Ser Tyr 165 170 175 tct cct ggc aca ctt gaa agt ccc ggt act ctt aat gca ttg gaa aca 933 Ser Pro Gly Thr Leu Glu Ser Pro Gly Thr Leu Asn Ala Leu Glu Thr 180 185 190 aat aat gtc tca cca gca gtt ata gag gaa gaa caa cat aca ctg tct 981 Asn Asn Val Ser Pro Ala Val Ile Glu Glu Glu Gln His Thr Leu Ser 195 200 205 ttg gaa gat ttg tca ttg tcc tta caa cac caa aat gaa aat gca aga 1029 Leu Glu Asp Leu Ser Leu Ser Leu Gln His Gln Asn Glu Asn Ala Arg 210 215 220 225 tta tct gca ccc cgc agt gca ccg cca cag gtt ccg act tca aag aca 1077 Leu Ser Ala Pro Arg Ser Ala Pro Pro Gln Val Pro Thr Ser Lys Thr 230 235 240 tcg tca ttt cac gat atg agt ctg gtt ata tct tca tca act tct gtg 1125 Ser Ser Phe His Asp Met Ser Leu Val Ile Ser Ser Ser Thr Ser Val 245 250 255 cat aag ata cca tca aat cca act tca act cga ggt tct cat tta tca 1173 His Lys Ile Pro Ser Asn Pro Thr Ser Thr Arg Gly Ser His Leu Ser 260 265 270 agt tac aaa tct aca ttg gac cct ggg aaa cct gca caa gca gca gca 1221 Ser Tyr Lys Ser Thr Leu Asp Pro Gly Lys Pro Ala Gln Ala Ala Ala 275 280 285 cca cca cca cca gaa ata gac att gac aat tta tta acc aaa agt gaa 1269 Pro Pro Pro Pro Glu Ile Asp Ile Asp Asn Leu Leu Thr Lys Ser Glu 290 295 300 305 ttg gat ctg gaa aca gac aca ttg agt agt gcc aca aat tct cca aac 1317 Leu Asp Leu Glu Thr Asp Thr Leu Ser Ser Ala Thr Asn Ser Pro Asn 310 315 320 ctt tta aga aat gat act tta caa gga att cca aca aga gat gac gaa 1365 Leu Leu Arg Asn Asp Thr Leu Gln Gly Ile Pro Thr Arg Asp Asp Glu 325 330 335 aat att gat gac ctg ccc cgt caa cta tca caa aat act agt gcg acg 1413 Asn Ile Asp Asp Leu Pro Arg Gln Leu Ser Gln Asn Thr Ser Ala Thr 340 345 350 tca aga aat act tcg gga aca tcg act tct aca gtg gtg aaa aat tca 1461 Ser Arg Asn Thr Ser Gly Thr Ser Thr Ser Thr Val Val Lys Asn Ser 355 360 365 aga tct ggt acg tca aaa tca acc tca acc tca act gct cat aac caa 1509 Arg Ser Gly Thr Ser Lys Ser Thr Ser Thr Ser Thr Ala His Asn Gln 370 375 380 385 aca gca gca att act cct ata atc ccg agt cac aac aag ttt cat caa 1557 Thr Ala Ala Ile Thr Pro Ile Ile Pro Ser His Asn Lys Phe His Gln 390 395 400 caa gtg ata aat acc aat gca aca aat agt tca tct tca cta gaa cca 1605 Gln Val Ile Asn Thr Asn Ala Thr Asn Ser Ser Ser Ser Leu Glu Pro 405 410 415 ttg ggg gtt ggc ata aat tca aat ctg tct cct aaa agt ggg aaa aag 1653 Leu Gly Val Gly Ile Asn Ser Asn Leu Ser Pro Lys Ser Gly Lys Lys 420 425 430 cgg aaa agt gga agt aaa gtc cga ggt gtg ttt tcg tca atg ttt ggg 1701 Arg Lys Ser Gly Ser Lys Val Arg Gly Val Phe Ser Ser Met Phe Gly 435 440 445 aaa aac aag tca acg tca tca tcg tcg tct tca aac tca ggt ctg aat 1749 Lys Asn Lys Ser Thr Ser Ser Ser Ser Ser Ser Asn Ser Gly Leu Asn 450 455 460 465 agc cac tca cag gaa gtc aat att aag atc agt act cca ttc aat gcc 1797 Ser His Ser Gln Glu Val Asn Ile Lys Ile Ser Thr Pro Phe Asn Ala 470 475 480 aag cac ctt gcc cat gtg ggc att gat gat aat ggt tca tac acc ggt 1845 Lys His Leu Ala His Val Gly Ile Asp Asp Asn Gly Ser Tyr Thr Gly 485 490 495 ttg cca ata gag tgg gaa aga tta tta tct gct agt ggt att acc aag 1893 Leu Pro Ile Glu Trp Glu Arg Leu Leu Ser Ala Ser Gly Ile Thr Lys 500 505 510 aag gaa caa caa cag cac cca caa gca gtg atg gat ata gtg gcg ttt 1941 Lys Glu Gln Gln Gln His Pro Gln Ala Val Met Asp Ile Val Ala Phe 515 520 525 tat caa gat aca agt gaa aac cct gat gac gct gca ttt aaa aag ttt 1989 Tyr Gln Asp Thr Ser Glu Asn Pro Asp Asp Ala Ala Phe Lys Lys Phe 530 535 540 545 cat ttt gat aat aat aaa agt agt tcg agt ggt tgg tct aat gaa aat 2037 His Phe Asp Asn Asn Lys Ser Ser Ser Ser Gly Trp Ser Asn Glu Asn 550 555 560 act cca cca gca aca ccg ggt ggg agt aac agt ggc agt ggc agt ggt 2085 Thr Pro Pro Ala Thr Pro Gly Gly Ser Asn Ser Gly Ser Gly Ser Gly 565 570 575 ggc ggt ggc gct cct tca agt ccc cat cgt aca cct cct tca tcg atc 2133 Gly Gly Gly Ala Pro Ser Ser Pro His Arg Thr Pro Pro Ser Ser Ile 580 585 590 att gaa aaa aac aac gtt gaa caa aaa gtg att acc cca tct cag tca 2181 Ile Glu Lys Asn Asn Val Glu Gln Lys Val Ile Thr Pro Ser Gln Ser 595 600 605 atg cca aca aag aca gag agt aaa cag ctg gaa aac cag cac cca cat 2229 Met Pro Thr Lys Thr Glu Ser Lys Gln Leu Glu Asn Gln His Pro His 610 615 620 625 gaa gat aat gct act cag tat aca cca aga aca cca aca tcc cat gta 2277 Glu Asp Asn Ala Thr Gln Tyr Thr Pro Arg Thr Pro Thr Ser His Val 630 635 640 caa gag ggt caa ttt att cca agt aga cca gct ccg aaa cca cca tca 2325 Gln Glu Gly Gln Phe Ile Pro Ser Arg Pro Ala Pro Lys Pro Pro Ser 645 650 655 aca ccg ctt tct tcc atg agt gtg tca cat aaa aca cct tct tcg caa 2373 Thr Pro Leu Ser Ser Met Ser Val Ser His Lys Thr Pro Ser Ser Gln 660 665 670 tca tta cca agg agt gat tca caa tcc gat att cgt tct tca acc cct 2421 Ser Leu Pro Arg Ser Asp Ser Gln Ser Asp Ile Arg Ser Ser Thr Pro 675 680 685 aaa tca cat caa gat gtt tcg cca agc aag atc aaa att cgt tca att 2469 Lys Ser His Gln Asp Val Ser Pro Ser Lys Ile Lys Ile Arg Ser Ile 690 695 700 705 tcg tca aaa tca tta aag tca atg cgg tct aga aaa agt ggg gat aag 2517 Ser Ser Lys Ser Leu Lys Ser Met Arg Ser Arg Lys Ser Gly Asp Lys 710 715 720 ttt act cat att gca cct gct cct cca cca cca tca tta cct tca att 2565 Phe Thr His Ile Ala Pro Ala Pro Pro Pro Pro Ser Leu Pro Ser Ile 725 730 735 cct aaa tca aag tcg cat tcg gca tct ttg tca agt caa ttg aga cca 2613 Pro Lys Ser Lys Ser His Ser Ala Ser Leu Ser Ser Gln Leu Arg Pro 740 745 750 gca aca aat gga tca aca act gcc cct att cca gca agt gcc gcg ttt 2661 Ala Thr Asn Gly Ser Thr Thr Ala Pro Ile Pro Ala Ser Ala Ala Phe 755 760 765 ggt ggt gag aat aat gct tta cca aaa caa aga ata aat gag ttc aag 2709 Gly Gly Glu Asn Asn Ala Leu Pro Lys Gln Arg Ile Asn Glu Phe Lys 770 775 780 785 gct cat aga gca cct cca cca cct cca ctg gca cca cct gca cca cct 2757 Ala His Arg Ala Pro Pro Pro Pro Pro Leu Ala Pro Pro Ala Pro Pro 790 795 800 gtg cct cct gct cca cca gcc aat tta tta tcg gaa cag act tct gag 2805 Val Pro Pro Ala Pro Pro Ala Asn Leu Leu Ser Glu Gln Thr Ser Glu 805 810 815 ata cct caa caa cgt act gct cct ctg caa gca tta gct gat gtt act 2853 Ile Pro Gln Gln Arg Thr Ala Pro Leu Gln Ala Leu Ala Asp Val Thr 820 825 830 gcc cca act aat att tat gaa att caa caa act aaa tat cag gaa gca 2901 Ala Pro Thr Asn Ile Tyr Glu Ile Gln Gln Thr Lys Tyr Gln Glu Ala 835 840 845 caa cag aaa tta cgt gag aag aag gct aga gaa ctt gaa gaa ata caa 2949 Gln Gln Lys Leu Arg Glu Lys Lys Ala Arg Glu Leu Glu Glu Ile Gln 850 855 860 865 aga cta cga gag aag aat gaa aga caa aat aga caa cag gag act ggg 2997 Arg Leu Arg Glu Lys Asn Glu Arg Gln Asn Arg Gln Gln Glu Thr Gly 870 875 880 caa aat aat gct gac acg gct agc ggt ggt agt aat att gct cca cca 3045 Gln Asn Asn Ala Asp Thr Ala Ser Gly Gly Ser Asn Ile Ala Pro Pro 885 890 895 gta cct gta cca aat aaa aaa ccg cct tct gga tct ggt ggt ggc cgt 3093 Val Pro Val Pro Asn Lys Lys Pro Pro Ser Gly Ser Gly Gly Gly Arg 900 905 910 gat gcc aaa caa gca gct ttg ata gcc caa aag aaa cga gaa gaa aag 3141 Asp Ala Lys Gln Ala Ala Leu Ile Ala Gln Lys Lys Arg Glu Glu Lys 915 920 925 aaa cgt aaa aac tta caa att att gcc aaa tta aag aca att tgt aat 3189 Lys Arg Lys Asn Leu Gln Ile Ile Ala Lys Leu Lys Thr Ile Cys Asn 930 935 940 945 cct gga gat cca aat gaa tta tat gtt gat tta gtt aaa att ggt caa 3237 Pro Gly Asp Pro Asn Glu Leu Tyr Val Asp Leu Val Lys Ile Gly Gln 950 955 960 ggt gcc tcc ggt gga gtt ttc ctt gct cat gat gtt cgt gat aaa tcc 3285 Gly Ala Ser Gly Gly Val Phe Leu Ala His Asp Val Arg Asp Lys Ser 965 970 975 aat att gtt gcc ata aaa caa atg aat tta gaa caa caa cct aaa aaa 3333 Asn Ile Val Ala Ile Lys Gln Met Asn Leu Glu Gln Gln Pro Lys Lys 980 985 990 gaa tta att att aat gaa att ttg gtt atg aaa ggt agt ctg cat ccc 3381 Glu Leu Ile Ile Asn Glu Ile Leu Val Met Lys Gly Ser Leu His Pro 995 1000 1005 aat att gtc aat ttt att gat tca tat ctt tta aaa ggt gat tta tgg 3429 Asn Ile Val Asn Phe Ile Asp Ser Tyr Leu Leu Lys Gly Asp Leu Trp 1010 1015 1020 1025 gtg att atg gaa tat atg gaa ggt gga tcc ctt acc gat ata gtg act 3477 Val Ile Met Glu Tyr Met Glu Gly Gly Ser Leu Thr Asp Ile Val Thr 1030 1035 1040 cat agt gtt atg acc gaa ggt caa att gga gtt gta tgt cgt gaa act 3525 His Ser Val Met Thr Glu Gly Gln Ile Gly Val Val Cys Arg Glu Thr 1045 1050 1055 ttg aaa ggt ctt aaa ttt tta cat tcc aaa ggg gtt atc cat cgt gat 3573 Leu Lys Gly Leu Lys Phe Leu His Ser Lys Gly Val Ile His Arg Asp 1060 1065 1070 att aaa tcc gat aat att tta tta aat atg gat ggt aac atc aag atc 3621 Ile Lys Ser Asp Asn Ile Leu Leu Asn Met Asp Gly Asn Ile Lys Ile 1075 1080 1085 act gat ttt ggg ttt tgt gct caa atc aat gaa atc aat ctg aaa cgt 3669 Thr Asp Phe Gly Phe Cys Ala Gln Ile Asn Glu Ile Asn Leu Lys Arg 1090 1095 1100 1105 atc act atg gtg ggt aca cca tat tgg atg gca cca gaa att gtt tca 3717 Ile Thr Met Val Gly Thr Pro Tyr Trp Met Ala Pro Glu Ile Val Ser 1110 1115 1120 cgt aaa gag tat ggt cca aaa gtt gat gtt tgg tca tta ggt atc atg 3765 Arg Lys Glu Tyr Gly Pro Lys Val Asp Val Trp Ser Leu Gly Ile Met 1125 1130 1135 att ata gaa atg tta gaa ggt gaa cca cca tat ttg aat gaa act cca 3813 Ile Ile Glu Met Leu Glu Gly Glu Pro Pro Tyr Leu Asn Glu Thr Pro 1140 1145 1150 ttg agg gca tta tat ctt att gca act aat ggt aca cca aaa tta aaa 3861 Leu Arg Ala Leu Tyr Leu Ile Ala Thr Asn Gly Thr Pro Lys Leu Lys 1155 1160 1165 gat cct gaa tct tta agt tat gat att aga aaa ttt ttg gca tgg tgt 3909 Asp Pro Glu Ser Leu Ser Tyr Asp Ile Arg Lys Phe Leu Ala Trp Cys 1170 1175 1180 1185 tta caa gtt gac ttt aat aaa aga gct gat gct gat gaa tta tta cat 3957 Leu Gln Val Asp Phe Asn Lys Arg Ala Asp Ala Asp Glu Leu Leu His 1190 1195 1200 gat aat ttt att act gaa tgt gat gat gta tcg tcg tta agt cca tta 4005 Asp Asn Phe Ile Thr Glu Cys Asp Asp Val Ser Ser Leu Ser Pro Leu 1205 1210 1215 gtg aaa att gct cga ttg aaa aaa atg agt gaa tct gat taatgaatgg 4054 Val Lys Ile Ala Arg Leu Lys Lys Met Ser Glu Ser Asp 1220 1225 1230 tggagttatc ctagaaataa aaacatttaa aaaaaaagaa gaagaacaac aagaacccta 4114 aattctactg ctgtcaatat attggctaat ttccattctc gtttctattt ctatttcgtt 4174 tttattcttt gaattattat tgttagtggt agagattttt actagtatat tttttttatt 4234 catttttata tttgtattta tatatatatt tttcatttag tatttactta cactgcagta 4294 tctttctttt ctgtgtagat gatatgtagt aataagttaa cttgttcaag acagtgaatg 4354 gaaatatatt atagcttgac tatataaggt ggagagctgt aattggcttt ccgtatagaa 4414 aagtcttgaa caaacgttac cagatttctg ctattcttat ttggtacgat tcgggcgtat 4474 gataggttta ttgagctc 4492 6 1230 PRT Candida albicans 6 Met Ser Ile Leu Ser Glu Asn Asn Pro Thr Pro Thr Ser Ile Thr Asp 1 5 10 15 Pro Asn Glu Ser Ser His Leu His Asn Pro Glu Leu Asn Ser Gly Thr 20 25 30 Arg Val Ala Ser Gly Pro Gly Pro Gly Pro Glu Val Glu Ser Thr Pro 35 40 45 Leu Ala Pro Pro Thr Glu Val Met Asn Thr Thr Ser Ala Asn Thr Ser 50 55 60 Ser Leu Ser Leu Gly Ser Pro Met His Glu Lys Ile Lys Gln Phe Asp 65 70 75 80 Gln Asp Glu Val Asp Thr Gly Glu Thr Asn Asp Arg Thr Ile Glu Ser 85 90 95 Gly Ser Ser Asp Ile Asp Asp Ser Gln Gln Ser His Asn Asn Asn Asn 100 105 110 Asn Asn Asn Asn Asn Asn Asn Glu Ser Asn Pro Glu Ser Ser Glu Gly 115 120 125 Asp Asp Glu Lys Thr Gln Gly Met Pro Pro Arg Met Pro Gly Thr Phe 130 135 140 Asn Val Lys Gly Leu His Gln Gly Asp Asp Ser Asp Asn Glu Lys Gln 145 150 155 160 Tyr Thr Glu Leu Thr Lys Ser Ile Asn Lys Arg Thr Ser Lys Asp Ser 165 170 175 Tyr Ser Pro Gly Thr Leu Glu Ser Pro Gly Thr Leu Asn Ala Leu Glu 180 185 190 Thr Asn Asn Val Ser Pro Ala Val Ile Glu Glu Glu Gln His Thr Leu 195 200 205 Ser Leu Glu Asp Leu Ser Leu Ser Leu Gln His Gln Asn Glu Asn Ala 210 215 220 Arg Leu Ser Ala Pro Arg Ser Ala Pro Pro Gln Val Pro Thr Ser Lys 225 230 235 240 Thr Ser Ser Phe His Asp Met Ser Leu Val Ile Ser Ser Ser Thr Ser 245 250 255 Val His Lys Ile Pro Ser Asn Pro Thr Ser Thr Arg Gly Ser His Leu 260 265 270 Ser Ser Tyr Lys Ser Thr Leu Asp Pro Gly Lys Pro Ala Gln Ala Ala 275 280 285 Ala Pro Pro Pro Pro Glu Ile Asp Ile Asp Asn Leu Leu Thr Lys Ser 290 295 300 Glu Leu Asp Leu Glu Thr Asp Thr Leu Ser Ser Ala Thr Asn Ser Pro 305 310 315 320 Asn Leu Leu Arg Asn Asp Thr Leu Gln Gly Ile Pro Thr Arg Asp Asp 325 330 335 Glu Asn Ile Asp Asp Leu Pro Arg Gln Leu Ser Gln Asn Thr Ser Ala 340 345 350 Thr Ser Arg Asn Thr Ser Gly Thr Ser Thr Ser Thr Val Val Lys Asn 355 360 365 Ser Arg Ser Gly Thr Ser Lys Ser Thr Ser Thr Ser Thr Ala His Asn 370 375 380 Gln Thr Ala Ala Ile Thr Pro Ile Ile Pro Ser His Asn Lys Phe His 385 390 395 400 Gln Gln Val Ile Asn Thr Asn Ala Thr Asn Ser Ser Ser Ser Leu Glu 405 410 415 Pro Leu Gly Val Gly Ile Asn Ser Asn Leu Ser Pro Lys Ser Gly Lys 420 425 430 Lys Arg Lys Ser Gly Ser Lys Val Arg Gly Val Phe Ser Ser Met Phe 435 440 445 Gly Lys Asn Lys Ser Thr Ser Ser Ser Ser Ser Ser Asn Ser Gly Leu 450 455 460 Asn Ser His Ser Gln Glu Val Asn Ile Lys Ile Ser Thr Pro Phe Asn 465 470 475 480 Ala Lys His Leu Ala His Val Gly Ile Asp Asp Asn Gly Ser Tyr Thr 485 490 495 Gly Leu Pro Ile Glu Trp Glu Arg Leu Leu Ser Ala Ser Gly Ile Thr 500 505 510 Lys Lys Glu Gln Gln Gln His Pro Gln Ala Val Met Asp Ile Val Ala 515 520 525 Phe Tyr Gln Asp Thr Ser Glu Asn Pro Asp Asp Ala Ala Phe Lys Lys 530 535 540 Phe His Phe Asp Asn Asn Lys Ser Ser Ser Ser Gly Trp Ser Asn Glu 545 550 555 560 Asn Thr Pro Pro Ala Thr Pro Gly Gly Ser Asn Ser Gly Ser Gly Ser 565 570 575 Gly Gly Gly Gly Ala Pro Ser Ser Pro His Arg Thr Pro Pro Ser Ser 580 585 590 Ile Ile Glu Lys Asn Asn Val Glu Gln Lys Val Ile Thr Pro Ser Gln 595 600 605 Ser Met Pro Thr Lys Thr Glu Ser Lys Gln Leu Glu Asn Gln His Pro 610 615 620 His Glu Asp Asn Ala Thr Gln Tyr Thr Pro Arg Thr Pro Thr Ser His 625 630 635 640 Val Gln Glu Gly Gln Phe Ile Pro Ser Arg Pro Ala Pro Lys Pro Pro 645 650 655 Ser Thr Pro Leu Ser Ser Met Ser Val Ser His Lys Thr Pro Ser Ser 660 665 670 Gln Ser Leu Pro Arg Ser Asp Ser Gln Ser Asp Ile Arg Ser Ser Thr 675 680 685 Pro Lys Ser His Gln Asp Val Ser Pro Ser Lys Ile Lys Ile Arg Ser 690 695 700 Ile Ser Ser Lys Ser Leu Lys Ser Met Arg Ser Arg Lys Ser Gly Asp 705 710 715 720 Lys Phe Thr His Ile Ala Pro Ala Pro Pro Pro Pro Ser Leu Pro Ser 725 730 735 Ile Pro Lys Ser Lys Ser His Ser Ala Ser Leu Ser Ser Gln Leu Arg 740 745 750 Pro Ala Thr Asn Gly Ser Thr Thr Ala Pro Ile Pro Ala Ser Ala Ala 755 760 765 Phe Gly Gly Glu Asn Asn Ala Leu Pro Lys Gln Arg Ile Asn Glu Phe 770 775 780 Lys Ala His Arg Ala Pro Pro Pro Pro Pro Leu Ala Pro Pro Ala Pro 785 790 795 800 Pro Val Pro Pro Ala Pro Pro Ala Asn Leu Leu Ser Glu Gln Thr Ser 805 810 815 Glu Ile Pro Gln Gln Arg Thr Ala Pro Leu Gln Ala Leu Ala Asp Val 820 825 830 Thr Ala Pro Thr Asn Ile Tyr Glu Ile Gln Gln Thr Lys Tyr Gln Glu 835 840 845 Ala Gln Gln Lys Leu Arg Glu Lys Lys Ala Arg Glu Leu Glu Glu Ile 850 855 860 Gln Arg Leu Arg Glu Lys Asn Glu Arg Gln Asn Arg Gln Gln Glu Thr 865 870 875 880 Gly Gln Asn Asn Ala Asp Thr Ala Ser Gly Gly Ser Asn Ile Ala Pro 885 890 895 Pro Val Pro Val Pro Asn Lys Lys Pro Pro Ser Gly Ser Gly Gly Gly 900 905 910 Arg Asp Ala Lys Gln Ala Ala Leu Ile Ala Gln Lys Lys Arg Glu Glu 915 920 925 Lys Lys Arg Lys Asn Leu Gln Ile Ile Ala Lys Leu Lys Thr Ile Cys 930 935 940 Asn Pro Gly Asp Pro Asn Glu Leu Tyr Val Asp Leu Val Lys Ile Gly 945 950 955 960 Gln Gly Ala Ser Gly Gly Val Phe Leu Ala His Asp Val Arg Asp Lys 965 970 975 Ser Asn Ile Val Ala Ile Lys Gln Met Asn Leu Glu Gln Gln Pro Lys 980 985 990 Lys Glu Leu Ile Ile Asn Glu Ile Leu Val Met Lys Gly Ser Leu His 995 1000 1005 Pro Asn Ile Val Asn Phe Ile Asp Ser Tyr Leu Leu Lys Gly Asp Leu 1010 1015 1020 Trp Val Ile Met Glu Tyr Met Glu Gly Gly Ser Leu Thr Asp Ile Val 1025 1030 1035 1040 Thr His Ser Val Met Thr Glu Gly Gln Ile Gly Val Val Cys Arg Glu 1045 1050 1055 Thr Leu Lys Gly Leu Lys Phe Leu His Ser Lys Gly Val Ile His Arg 1060 1065 1070 Asp Ile Lys Ser Asp Asn Ile Leu Leu Asn Met Asp Gly Asn Ile Lys 1075 1080 1085 Ile Thr Asp Phe Gly Phe Cys Ala Gln Ile Asn Glu Ile Asn Leu Lys 1090 1095 1100 Arg Ile Thr Met Val Gly Thr Pro Tyr Trp Met Ala Pro Glu Ile Val 1105 1110 1115 1120 Ser Arg Lys Glu Tyr Gly Pro Lys Val Asp Val Trp Ser Leu Gly Ile 1125 1130 1135 Met Ile Ile Glu Met Leu Glu Gly Glu Pro Pro Tyr Leu Asn Glu Thr 1140 1145 1150 Pro Leu Arg Ala Leu Tyr Leu Ile Ala Thr Asn Gly Thr Pro Lys Leu 1155 1160 1165 Lys Asp Pro Glu Ser Leu Ser Tyr Asp Ile Arg Lys Phe Leu Ala Trp 1170 1175 1180 Cys Leu Gln Val Asp Phe Asn Lys Arg Ala Asp Ala Asp Glu Leu Leu 1185 1190 1195 1200 His Asp Asn Phe Ile Thr Glu Cys Asp Asp Val Ser Ser Leu Ser Pro 1205 1210 1215 Leu Val Lys Ile Ala Arg Leu Lys Lys Met Ser Glu Ser Asp 1220 1225 1230 7 3496 DNA Candida albicans CDS (432)...(3344) 7 gaattctttt tagaagagaa agaaaaaatt cccaaaaaaa aaagatttca tttaattcca 60 cgggaacatt gattacaacc acgtcaacag tttccctttt atattgaaat caacattcaa 120 ttttgtcttt tttttttttt cattgatttt tccccaatct ttttatcttc atattaatat 180 tggatatcaa ttactaatac tgtcagggat agtttagtaa atatttacat tctccattca 240 atcctaaatt tttttttata tagctagttt ttggttgaaa aaaaaaaaat agggggaagg 300 aagttttttt ttctatttat ttaattgttt tgattccaac catattgtat atttgtcttg 360 tcagttatat tactttcttg ttacttaatt attaattatt tgctatatta ttgaattgaa 420 tcctcaaaag a atg aca agt att tat aca tca gat ttg aaa aac cat aga 470 Met Thr Ser Ile Tyr Thr Ser Asp Leu Lys Asn His Arg 1 5 10 cgt gcg cca cct cca cca aat ggg gca gct ggc tct ggc tca ggt tct 518 Arg Ala Pro Pro Pro Pro Asn Gly Ala Ala Gly Ser Gly Ser Gly Ser 15 20 25 ggc tca ggt tct ggt tct ggt tct ggc agt ttg gct aat att gtt acc 566 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Leu Ala Asn Ile Val Thr 30 35 40 45 agt tct aat agt ctt ggc gta aca gca aat caa acc aaa cct att caa 614 Ser Ser Asn Ser Leu Gly Val Thr Ala Asn Gln Thr Lys Pro Ile Gln 50 55 60 tta aat ata aat tct agc aaa cgt caa tca ggt tgg gtt cat gtt aaa 662 Leu Asn Ile Asn Ser Ser Lys Arg Gln Ser Gly Trp Val His Val Lys 65 70 75 gat gat ggt att ttc aca tca ttt aga tgg aac aaa cgg ttt atg gtt 710 Asp Asp Gly Ile Phe Thr Ser Phe Arg Trp Asn Lys Arg Phe Met Val 80 85 90 att aat gat aaa act tta aac ttt tat aaa caa gaa cca tat tct agt 758 Ile Asn Asp Lys Thr Leu Asn Phe Tyr Lys Gln Glu Pro Tyr Ser Ser 95 100 105 gat ggt aat tcc aat tct aat acc cct gat tta tca ttc cca cta tat 806 Asp Gly Asn Ser Asn Ser Asn Thr Pro Asp Leu Ser Phe Pro Leu Tyr 110 115 120 125 tta att aat aat att aat ttg aaa cca aac tcc ggg tat agc aaa act 854 Leu Ile Asn Asn Ile Asn Leu Lys Pro Asn Ser Gly Tyr Ser Lys Thr 130 135 140 tca caa tca ttt gaa att gtt ccc aaa aac aat aat aaa tca att ttg 902 Ser Gln Ser Phe Glu Ile Val Pro Lys Asn Asn Asn Lys Ser Ile Leu 145 150 155 att tct gtt aaa acc aat aat gat tat ttg gat tgg cta gat gca ttc 950 Ile Ser Val Lys Thr Asn Asn Asp Tyr Leu Asp Trp Leu Asp Ala Phe 160 165 170 acc aca aaa tgt cct tta gta caa att ggt gaa aat aat agt ggt gta 998 Thr Thr Lys Cys Pro Leu Val Gln Ile Gly Glu Asn Asn Ser Gly Val 175 180 185 tca agt agt cac cct cat tta caa att caa cat tta acc aat ggt tcc 1046 Ser Ser Ser His Pro His Leu Gln Ile Gln His Leu Thr Asn Gly Ser 190 195 200 205 ttg aac ggc aac tca tct tca tca cca aca tct gga tta tta tct tct 1094 Leu Asn Gly Asn Ser Ser Ser Ser Pro Thr Ser Gly Leu Leu Ser Ser 210 215 220 tca gtg cta act gga ggt aat tct ggc gtt tct ggt cct att aat ttc 1142 Ser Val Leu Thr Gly Gly Asn Ser Gly Val Ser Gly Pro Ile Asn Phe 225 230 235 act cat aaa gta cac gtg gga ttt gat cct gcc agt ggt aat ttt act 1190 Thr His Lys Val His Val Gly Phe Asp Pro Ala Ser Gly Asn Phe Thr 240 245 250 gga tta cca gac act tgg aaa agt tta tta caa cat tcg aaa atc act 1238 Gly Leu Pro Asp Thr Trp Lys Ser Leu Leu Gln His Ser Lys Ile Thr 255 260 265 aat gag gat tgg aaa aaa gat cct gtt gct gtt att gaa gtt tta gaa 1286 Asn Glu Asp Trp Lys Lys Asp Pro Val Ala Val Ile Glu Val Leu Glu 270 275 280 285 ttt tat tcc gat ata aat gga ggt aat tca gct gct gga act cca att 1334 Phe Tyr Ser Asp Ile Asn Gly Gly Asn Ser Ala Ala Gly Thr Pro Ile 290 295 300 gga tca ccc atg atc aat tcc aaa acc aac aat aat aat aat gac cct 1382 Gly Ser Pro Met Ile Asn Ser Lys Thr Asn Asn Asn Asn Asn Asp Pro 305 310 315 aac aat tac tca tca acc aaa aac aat gtc caa gag gca aat tta caa 1430 Asn Asn Tyr Ser Ser Thr Lys Asn Asn Val Gln Glu Ala Asn Leu Gln 320 325 330 gaa tgg gta aaa cct cca gca aaa tct act gtc tca caa ttc aaa cct 1478 Glu Trp Val Lys Pro Pro Ala Lys Ser Thr Val Ser Gln Phe Lys Pro 335 340 345 agt cga gct gca cca aaa cca cca act cca tat cat ttg aca caa cta 1526 Ser Arg Ala Ala Pro Lys Pro Pro Thr Pro Tyr His Leu Thr Gln Leu 350 355 360 365 aat ggc tct tcc cac caa cat aca tca tca tca ggc tca tta cct agt 1574 Asn Gly Ser Ser His Gln His Thr Ser Ser Ser Gly Ser Leu Pro Ser 370 375 380 tct ggt aat aat aat aat aat aac agc act aac aat aat aat act aaa 1622 Ser Gly Asn Asn Asn Asn Asn Asn Ser Thr Asn Asn Asn Asn Thr Lys 385 390 395 aac gtt tca cca ttg aat aat ttg atg aat aaa tct gaa ctt att cct 1670 Asn Val Ser Pro Leu Asn Asn Leu Met Asn Lys Ser Glu Leu Ile Pro 400 405 410 gct aga aga gct cca cca cct cca aca agt ggc aca tct tca gat aca 1718 Ala Arg Arg Ala Pro Pro Pro Pro Thr Ser Gly Thr Ser Ser Asp Thr 415 420 425 tat tct aat aag aat cat caa gat aga tct gga tat gaa caa caa cgt 1766 Tyr Ser Asn Lys Asn His Gln Asp Arg Ser Gly Tyr Glu Gln Gln Arg 430 435 440 445 caa caa cgt act gac tca tca caa caa caa caa caa caa aag caa cat 1814 Gln Gln Arg Thr Asp Ser Ser Gln Gln Gln Gln Gln Gln Lys Gln His 450 455 460 caa tat caa cag aaa tcc caa caa caa caa caa caa cca caa caa cca 1862 Gln Tyr Gln Gln Lys Ser Gln Gln Gln Gln Gln Gln Pro Gln Gln Pro 465 470 475 tta tct ctg cat caa ggt ggg act tcg cat att ccg aaa caa gta cct 1910 Leu Ser Leu His Gln Gly Gly Thr Ser His Ile Pro Lys Gln Val Pro 480 485 490 cct aca tta cca tca tct gga cca ccc act cag gct gct tca gga aaa 1958 Pro Thr Leu Pro Ser Ser Gly Pro Pro Thr Gln Ala Ala Ser Gly Lys 495 500 505 tca atg cca tct aaa att cat cct gat ctt aag att caa caa ggc aca 2006 Ser Met Pro Ser Lys Ile His Pro Asp Leu Lys Ile Gln Gln Gly Thr 510 515 520 525 aat aat tat att aag agt agc ggt act gat gct aat caa gtc gat ggt 2054 Asn Asn Tyr Ile Lys Ser Ser Gly Thr Asp Ala Asn Gln Val Asp Gly 530 535 540 gat gct aaa caa ttt att aaa cca ttt aat tta caa ctg aaa aag agt 2102 Asp Ala Lys Gln Phe Ile Lys Pro Phe Asn Leu Gln Leu Lys Lys Ser 545 550 555 cag caa caa ttg gca tca aaa caa ccg tca cca cct tca tct caa caa 2150 Gln Gln Gln Leu Ala Ser Lys Gln Pro Ser Pro Pro Ser Ser Gln Gln 560 565 570 cag caa caa aaa cct atg aca tca cat gga tta atg ggt aca tca cat 2198 Gln Gln Gln Lys Pro Met Thr Ser His Gly Leu Met Gly Thr Ser His 575 580 585 tca gtt act aaa cca ttg aat cca gtc aat gat cca atc aaa cca tta 2246 Ser Val Thr Lys Pro Leu Asn Pro Val Asn Asp Pro Ile Lys Pro Leu 590 595 600 605 aat ttg aaa tca tct aaa tcc aaa gaa gca tta aat gaa act ctg ggg 2294 Asn Leu Lys Ser Ser Lys Ser Lys Glu Ala Leu Asn Glu Thr Leu Gly 610 615 620 gtg ctg aaa aca cca tca cct aca gat aaa tca aat aaa cca act gca 2342 Val Leu Lys Thr Pro Ser Pro Thr Asp Lys Ser Asn Lys Pro Thr Ala 625 630 635 cct gct agt ggt cct gca gtg acc aaa aca gct aaa caa ctc aag aag 2390 Pro Ala Ser Gly Pro Ala Val Thr Lys Thr Ala Lys Gln Leu Lys Lys 640 645 650 gaa cga gaa aga ttg aat gat tta caa atc att gct aaa ttg aaa aca 2438 Glu Arg Glu Arg Leu Asn Asp Leu Gln Ile Ile Ala Lys Leu Lys Thr 655 660 665 gtg gtt aat aat caa gat cct aaa cca tta ttt aga att gtt gaa aaa 2486 Val Val Asn Asn Gln Asp Pro Lys Pro Leu Phe Arg Ile Val Glu Lys 670 675 680 685 gct ggt caa ggt gct agt ggg aat gtt tat ttg gcg gaa atg atc aaa 2534 Ala Gly Gln Gly Ala Ser Gly Asn Val Tyr Leu Ala Glu Met Ile Lys 690 695 700 gat aat aat cga aag att gcg att aaa caa atg gat ctt gat gct caa 2582 Asp Asn Asn Arg Lys Ile Ala Ile Lys Gln Met Asp Leu Asp Ala Gln 705 710 715 ccc cgt aaa gag tta ata ata aat gaa atc ttg gtt atg aaa gat agt 2630 Pro Arg Lys Glu Leu Ile Ile Asn Glu Ile Leu Val Met Lys Asp Ser 720 725 730 caa cat aaa aat att gtt aat ttt ttg gat tct tat tta att ggt gat 2678 Gln His Lys Asn Ile Val Asn Phe Leu Asp Ser Tyr Leu Ile Gly Asp 735 740 745 aat gaa tta tgg gta att atg gaa tat atg caa ggt ggt tca tta acg 2726 Asn Glu Leu Trp Val Ile Met Glu Tyr Met Gln Gly Gly Ser Leu Thr 750 755 760 765 gaa atc att gaa aat aat gat ttt aaa ttg aat gaa aaa caa att gcc 2774 Glu Ile Ile Glu Asn Asn Asp Phe Lys Leu Asn Glu Lys Gln Ile Ala 770 775 780 act ata tgt ttt gaa acc tta aag ggg tta caa cat tta cat aaa aaa 2822 Thr Ile Cys Phe Glu Thr Leu Lys Gly Leu Gln His Leu His Lys Lys 785 790 795 cat att att cat cgt gat att aaa tcc gat aat gtt tta tta gat gca 2870 His Ile Ile His Arg Asp Ile Lys Ser Asp Asn Val Leu Leu Asp Ala 800 805 810 tat ggt aat gtt aaa atc act gat ttt gga ttt tgt gct aaa tta act 2918 Tyr Gly Asn Val Lys Ile Thr Asp Phe Gly Phe Cys Ala Lys Leu Thr 815 820 825 gat caa aga aat aaa cgt gcc aca atg gtg ggg aca cca tat tgg atg 2966 Asp Gln Arg Asn Lys Arg Ala Thr Met Val Gly Thr Pro Tyr Trp Met 830 835 840 845 gca cct gaa gtg gtt aaa caa aag gaa tat gat gaa aaa gtt gat gtt 3014 Ala Pro Glu Val Val Lys Gln Lys Glu Tyr Asp Glu Lys Val Asp Val 850 855 860 tgg tca ttg ggg att atg act att gaa atg att gaa gga gaa cca cct 3062 Trp Ser Leu Gly Ile Met Thr Ile Glu Met Ile Glu Gly Glu Pro Pro 865 870 875 tat ttg aat gaa gaa cca tta aaa gct tta tat ctt ata gct act aat 3110 Tyr Leu Asn Glu Glu Pro Leu Lys Ala Leu Tyr Leu Ile Ala Thr Asn 880 885 890 ggt aca cca aaa ttg aaa aaa ccc gaa tta tta tcg aat tca att aaa 3158 Gly Thr Pro Lys Leu Lys Lys Pro Glu Leu Leu Ser Asn Ser Ile Lys 895 900 905 aaa ttc tta tca att tgt ctt tgt gtt gat gtt aga tat cgt gct agt 3206 Lys Phe Leu Ser Ile Cys Leu Cys Val Asp Val Arg Tyr Arg Ala Ser 910 915 920 925 act gat gaa tta tta gaa cat tca ttt att caa cat aaa tca ggg aaa 3254 Thr Asp Glu Leu Leu Glu His Ser Phe Ile Gln His Lys Ser Gly Lys 930 935 940 att gaa gaa ttg gca cca tta tta gaa tgg aaa aaa caa caa caa aag 3302 Ile Glu Glu Leu Ala Pro Leu Leu Glu Trp Lys Lys Gln Gln Gln Lys 945 950 955 cat caa cag cat aaa caa gaa aca ctg gat aca gga ttt gca 3344 His Gln Gln His Lys Gln Glu Thr Leu Asp Thr Gly Phe Ala 960 965 970 tagagattga atatagccgt agaaaactgg tactttggtt ttggtataat atatttatgt 3404 gatgtgttgt gtgtatggtt agtttagatt tggattttta gttttttaga gtttagtttt 3464 tcaattttta gttttagaga caatattcta ga 3496 8 971 PRT Candida albicans 8 Met Thr Ser Ile Tyr Thr Ser Asp Leu Lys Asn His Arg Arg Ala Pro 1 5 10 15 Pro Pro Pro Asn Gly Ala Ala Gly Ser Gly Ser Gly Ser Gly Ser Gly 20 25 30 Ser Gly Ser Gly Ser Gly Ser Leu Ala Asn Ile Val Thr Ser Ser Asn 35 40 45 Ser Leu Gly Val Thr Ala Asn Gln Thr Lys Pro Ile Gln Leu Asn Ile 50 55 60 Asn Ser Ser Lys Arg Gln Ser Gly Trp Val His Val Lys Asp Asp Gly 65 70 75 80 Ile Phe Thr Ser Phe Arg Trp Asn Lys Arg Phe Met Val Ile Asn Asp 85 90 95 Lys Thr Leu Asn Phe Tyr Lys Gln Glu Pro Tyr Ser Ser Asp Gly Asn 100 105 110 Ser Asn Ser Asn Thr Pro Asp Leu Ser Phe Pro Leu Tyr Leu Ile Asn 115 120 125 Asn Ile Asn Leu Lys Pro Asn Ser Gly Tyr Ser Lys Thr Ser Gln Ser 130 135 140 Phe Glu Ile Val Pro Lys Asn Asn Asn Lys Ser Ile Leu Ile Ser Val 145 150 155 160 Lys Thr Asn Asn Asp Tyr Leu Asp Trp Leu Asp Ala Phe Thr Thr Lys 165 170 175 Cys Pro Leu Val Gln Ile Gly Glu Asn Asn Ser Gly Val Ser Ser Ser 180 185 190 His Pro His Leu Gln Ile Gln His Leu Thr Asn Gly Ser Leu Asn Gly 195 200 205 Asn Ser Ser Ser Ser Pro Thr Ser Gly Leu Leu Ser Ser Ser Val Leu 210 215 220 Thr Gly Gly Asn Ser Gly Val Ser Gly Pro Ile Asn Phe Thr His Lys 225 230 235 240 Val His Val Gly Phe Asp Pro Ala Ser Gly Asn Phe Thr Gly Leu Pro 245 250 255 Asp Thr Trp Lys Ser Leu Leu Gln His Ser Lys Ile Thr Asn Glu Asp 260 265 270 Trp Lys Lys Asp Pro Val Ala Val Ile Glu Val Leu Glu Phe Tyr Ser 275 280 285 Asp Ile Asn Gly Gly Asn Ser Ala Ala Gly Thr Pro Ile Gly Ser Pro 290 295 300 Met Ile Asn Ser Lys Thr Asn Asn Asn Asn Asn Asp Pro Asn Asn Tyr 305 310 315 320 Ser Ser Thr Lys Asn Asn Val Gln Glu Ala Asn Leu Gln Glu Trp Val 325 330 335 Lys Pro Pro Ala Lys Ser Thr Val Ser Gln Phe Lys Pro Ser Arg Ala 340 345 350 Ala Pro Lys Pro Pro Thr Pro Tyr His Leu Thr Gln Leu Asn Gly Ser 355 360 365 Ser His Gln His Thr Ser Ser Ser Gly Ser Leu Pro Ser Ser Gly Asn 370 375 380 Asn Asn Asn Asn Asn Ser Thr Asn Asn Asn Asn Thr Lys Asn Val Ser 385 390 395 400 Pro Leu Asn Asn Leu Met Asn Lys Ser Glu Leu Ile Pro Ala Arg Arg 405 410 415 Ala Pro Pro Pro Pro Thr Ser Gly Thr Ser Ser Asp Thr Tyr Ser Asn 420 425 430 Lys Asn His Gln Asp Arg Ser Gly Tyr Glu Gln Gln Arg Gln Gln Arg 435 440 445 Thr Asp Ser Ser Gln Gln Gln Gln Gln Gln Lys Gln His Gln Tyr Gln 450 455 460 Gln Lys Ser Gln Gln Gln Gln Gln Gln Pro Gln Gln Pro Leu Ser Leu 465 470 475 480 His Gln Gly Gly Thr Ser His Ile Pro Lys Gln Val Pro Pro Thr Leu 485 490 495 Pro Ser Ser Gly Pro Pro Thr Gln Ala Ala Ser Gly Lys Ser Met Pro 500 505 510 Ser Lys Ile His Pro Asp Leu Lys Ile Gln Gln Gly Thr Asn Asn Tyr 515 520 525 Ile Lys Ser Ser Gly Thr Asp Ala Asn Gln Val Asp Gly Asp Ala Lys 530 535 540 Gln Phe Ile Lys Pro Phe Asn Leu Gln Leu Lys Lys Ser Gln Gln Gln 545 550 555 560 Leu Ala Ser Lys Gln Pro Ser Pro Pro Ser Ser Gln Gln Gln Gln Gln 565 570 575 Lys Pro Met Thr Ser His Gly Leu Met Gly Thr Ser His Ser Val Thr 580 585 590 Lys Pro Leu Asn Pro Val Asn Asp Pro Ile Lys Pro Leu Asn Leu Lys 595 600 605 Ser Ser Lys Ser Lys Glu Ala Leu Asn Glu Thr Leu Gly Val Leu Lys 610 615 620 Thr Pro Ser Pro Thr Asp Lys Ser Asn Lys Pro Thr Ala Pro Ala Ser 625 630 635 640 Gly Pro Ala Val Thr Lys Thr Ala Lys Gln Leu Lys Lys Glu Arg Glu 645 650 655 Arg Leu Asn Asp Leu Gln Ile Ile Ala Lys Leu Lys Thr Val Val Asn 660 665 670 Asn Gln Asp Pro Lys Pro Leu Phe Arg Ile Val Glu Lys Ala Gly Gln 675 680 685 Gly Ala Ser Gly Asn Val Tyr Leu Ala Glu Met Ile Lys Asp Asn Asn 690 695 700 Arg Lys Ile Ala Ile Lys Gln Met Asp Leu Asp Ala Gln Pro Arg Lys 705 710 715 720 Glu Leu Ile Ile Asn Glu Ile Leu Val Met Lys Asp Ser Gln His Lys 725 730 735 Asn Ile Val Asn Phe Leu Asp Ser Tyr Leu Ile Gly Asp Asn Glu Leu 740 745 750 Trp Val Ile Met Glu Tyr Met Gln Gly Gly Ser Leu Thr Glu Ile Ile 755 760 765 Glu Asn Asn Asp Phe Lys Leu Asn Glu Lys Gln Ile Ala Thr Ile Cys 770 775 780 Phe Glu Thr Leu Lys Gly Leu Gln His Leu His Lys Lys His Ile Ile 785 790 795 800 His Arg Asp Ile Lys Ser Asp Asn Val Leu Leu Asp Ala Tyr Gly Asn 805 810 815 Val Lys Ile Thr Asp Phe Gly Phe Cys Ala Lys Leu Thr Asp Gln Arg 820 825 830 Asn Lys Arg Ala Thr Met Val Gly Thr Pro Tyr Trp Met Ala Pro Glu 835 840 845 Val Val Lys Gln Lys Glu Tyr Asp Glu Lys Val Asp Val Trp Ser Leu 850 855 860 Gly Ile Met Thr Ile Glu Met Ile Glu Gly Glu Pro Pro Tyr Leu Asn 865 870 875 880 Glu Glu Pro Leu Lys Ala Leu Tyr Leu Ile Ala Thr Asn Gly Thr Pro 885 890 895 Lys Leu Lys Lys Pro Glu Leu Leu Ser Asn Ser Ile Lys Lys Phe Leu 900 905 910 Ser Ile Cys Leu Cys Val Asp Val Arg Tyr Arg Ala Ser Thr Asp Glu 915 920 925 Leu Leu Glu His Ser Phe Ile Gln His Lys Ser Gly Lys Ile Glu Glu 930 935 940 Leu Ala Pro Leu Leu Glu Trp Lys Lys Gln Gln Gln Lys His Gln Gln 945 950 955 960 His Lys Gln Glu Thr Leu Asp Thr Gly Phe Ala 965 970 9 1031 DNA Candida albicans CDS (271)...(843) 9 caaccaaacc aactttcatc cttctaccaa tatcttcaac aaaagtttta ttcaatacta 60 ttttaaaaat aacagtgtta ctcgttcatt tgatttgtta ataagactga tttacccact 120 ttttagttcc tataatcata cagatttctc gtcctaaatc tatttttatt gttattttta 180 ctttagtttt cacttttgct ttcagttttt tcttttttta gcacaagaga aaagtattca 240 gctcataaat aattaatata tccatatatc atg caa act ata aaa tgt gtt gtt 294 Met Gln Thr Ile Lys Cys Val Val 1 5 gtc ggt gat ggt gcc gtt ggt aaa act tgc tta tta atc tcg tat acc 342 Val Gly Asp Gly Ala Val Gly Lys Thr Cys Leu Leu Ile Ser Tyr Thr 10 15 20 act agt aaa ttt cca gct gat tat gtt cct act gtt ttt gat aat tat 390 Thr Ser Lys Phe Pro Ala Asp Tyr Val Pro Thr Val Phe Asp Asn Tyr 25 30 35 40 gct gta acc gtg atg ata gga gac gaa cca ttt acc ttg gga tta ttt 438 Ala Val Thr Val Met Ile Gly Asp Glu Pro Phe Thr Leu Gly Leu Phe 45 50 55 gat act gct ggt caa gaa gat tac gac aga tta agg cct ttg tca tat 486 Asp Thr Ala Gly Gln Glu Asp Tyr Asp Arg Leu Arg Pro Leu Ser Tyr 60 65 70 cca tcg act gat gta ttc ctt gtt tgt ttt tcc gtc att tct ccc gct 534 Pro Ser Thr Asp Val Phe Leu Val Cys Phe Ser Val Ile Ser Pro Ala 75 80 85 tcg ttt gaa aat gtt aaa gaa aaa tgg ttc cca gaa gtt cat cac cat 582 Ser Phe Glu Asn Val Lys Glu Lys Trp Phe Pro Glu Val His His His 90 95 100 tgt ccc ggt gtg cca ata att att gtc ggt acc caa act gat tta cga 630 Cys Pro Gly Val Pro Ile Ile Ile Val Gly Thr Gln Thr Asp Leu Arg 105 110 115 120 aac gat gat gtt att tta cag aga ttg cac aga caa aaa ttg tcc cca 678 Asn Asp Asp Val Ile Leu Gln Arg Leu His Arg Gln Lys Leu Ser Pro 125 130 135 atc acc cag gaa cag ggt gaa aaa ttg gct aag gaa ttg aga gct gtc 726 Ile Thr Gln Glu Gln Gly Glu Lys Leu Ala Lys Glu Leu Arg Ala Val 140 145 150 aag tat gtt gag tgt tct gca ttg act caa aga gga ttg aaa aca gtg 774 Lys Tyr Val Glu Cys Ser Ala Leu Thr Gln Arg Gly Leu Lys Thr Val 155 160 165 ttt gac gag gct ata gta gct gca tta gaa cct cct gta att aaa aaa 822 Phe Asp Glu Ala Ile Val Ala Ala Leu Glu Pro Pro Val Ile Lys Lys 170 175 180 tcg aaa aag tgt act att tta taggtcggcg atactagaag atagaggata 873 Ser Lys Lys Cys Thr Ile Leu 185 190 ttggaaatag ggcatacatg agatattgaa tatctatcat taaatatata attagttttt 933 ttctaaaacc tatctttagg tttgatctcg tttgatgtgt tgggctgttt cgcaaaacag 993 tgttccaatc aataaaaaga tgtgtgtaag actctaga 1031 10 191 PRT Candida albicans 10 Met Gln Thr Ile Lys Cys Val Val Val Gly Asp Gly Ala Val Gly Lys 1 5 10 15 Thr Cys Leu Leu Ile Ser Tyr Thr Thr Ser Lys Phe Pro Ala Asp Tyr 20 25 30 Val Pro Thr Val Phe Asp Asn Tyr Ala Val Thr Val Met Ile Gly Asp 35 40 45 Glu Pro Phe Thr Leu Gly Leu Phe Asp Thr Ala Gly Gln Glu Asp Tyr 50 55 60 Asp Arg Leu Arg Pro Leu Ser Tyr Pro Ser Thr Asp Val Phe Leu Val 65 70 75 80 Cys Phe Ser Val Ile Ser Pro Ala Ser Phe Glu Asn Val Lys Glu Lys 85 90 95 Trp Phe Pro Glu Val His His His Cys Pro Gly Val Pro Ile Ile Ile 100 105 110 Val Gly Thr Gln Thr Asp Leu Arg Asn Asp Asp Val Ile Leu Gln Arg 115 120 125 Leu His Arg Gln Lys Leu Ser Pro Ile Thr Gln Glu Gln Gly Glu Lys 130 135 140 Leu Ala Lys Glu Leu Arg Ala Val Lys Tyr Val Glu Cys Ser Ala Leu 145 150 155 160 Thr Gln Arg Gly Leu Lys Thr Val Phe Asp Glu Ala Ile Val Ala Ala 165 170 175 Leu Glu Pro Pro Val Ile Lys Lys Ser Lys Lys Cys Thr Ile Leu 180 185 190 11 2231 DNA Candida albicans CDS (291)...(2195) 11 aagcttgttt cttatctcct tagtatattg ttttacaaca ccacatacac atacacatat 60 agccttcatt agccttcatt ttgacatatt tcaataacaa tcaagaacta caagtcataa 120 ctgacacaca tataatatct taattgttat tataaattta ttcttgatta gattttagac 180 gggcagaaac aaaaacggaa aatccaactc atccccgata actacacaca tctatattaa 240 atcatctatt agtctatcag ttatatctcc ctcccctttt cttctaacaa atg att 296 Met Ile 1 aag acg ttt cgg aaa agt aaa aga ctg tcg agt aat tca agt tca ccc 344 Lys Thr Phe Arg Lys Ser Lys Arg Leu Ser Ser Asn Ser Ser Ser Pro 5 10 15 aag aaa aca ata tct cga gta tca tca act tca agt aat caa aca tct 392 Lys Lys Thr Ile Ser Arg Val Ser Ser Thr Ser Ser Asn Gln Thr Ser 20 25 30 cat gat gga ata tta caa tca cct aaa aaa gtc att aga gct cta tat 440 His Asp Gly Ile Leu Gln Ser Pro Lys Lys Val Ile Arg Ala Leu Tyr 35 40 45 50 gat tat gaa cct caa ggt cct gga gaa ttg aaa ttt ttc aaa gga gat 488 Asp Tyr Glu Pro Gln Gly Pro Gly Glu Leu Lys Phe Phe Lys Gly Asp 55 60 65 ttt ttc cat gta tta aat gat gtt gat gat gaa tta cat aaa gaa gcg 536 Phe Phe His Val Leu Asn Asp Val Asp Asp Glu Leu His Lys Glu Ala 70 75 80 gaa cgt aat gga tgg ata gaa gca aca aat cca atg act caa ctt aaa 584 Glu Arg Asn Gly Trp Ile Glu Ala Thr Asn Pro Met Thr Gln Leu Lys 85 90 95 ggg atg gtc ccc att agt tat ttt gaa ata ttt gat cga tct cgt cct 632 Gly Met Val Pro Ile Ser Tyr Phe Glu Ile Phe Asp Arg Ser Arg Pro 100 105 110 aca gtt aca gca tca tca aac agt ttt aca aat tcc att gat att caa 680 Thr Val Thr Ala Ser Ser Asn Ser Phe Thr Asn Ser Ile Asp Ile Gln 115 120 125 130 cat caa cat caa caa gga att cac aat gga aca gga aat cga aat tta 728 His Gln His Gln Gln Gly Ile His Asn Gly Thr Gly Asn Arg Asn Leu 135 140 145 aat caa aca tta tat gct gtt aca cta tat gaa ttt aaa gct gaa cga 776 Asn Gln Thr Leu Tyr Ala Val Thr Leu Tyr Glu Phe Lys Ala Glu Arg 150 155 160 gat gat gaa ttg gat ata atg cct aat gaa aat tta att att tgt gca 824 Asp Asp Glu Leu Asp Ile Met Pro Asn Glu Asn Leu Ile Ile Cys Ala 165 170 175 cat cat gat tat gaa tgg ttt att gcc aaa cca ata aat cga tta ggt 872 His His Asp Tyr Glu Trp Phe Ile Ala Lys Pro Ile Asn Arg Leu Gly 180 185 190 gga cca ggt tta gta cct gtt tct tat gtt aaa ata att gat ctt tta 920 Gly Pro Gly Leu Val Pro Val Ser Tyr Val Lys Ile Ile Asp Leu Leu 195 200 205 210 aac cct aat tct cat tat aca tca att gat aca tca agg cga tca caa 968 Asn Pro Asn Ser His Tyr Thr Ser Ile Asp Thr Ser Arg Arg Ser Gln 215 220 225 gtc ata caa gta atc aat gga ttt aat ata ccg aca gta gaa caa tgg 1016 Val Ile Gln Val Ile Asn Gly Phe Asn Ile Pro Thr Val Glu Gln Trp 230 235 240 aaa aat caa act gcc aaa tat caa gct tca aca atc ccc ctt ggt tca 1064 Lys Asn Gln Thr Ala Lys Tyr Gln Ala Ser Thr Ile Pro Leu Gly Ser 245 250 255 ata tca gga agt ggt act cca cca aca tca gct aat tca caa tat ttt 1112 Ile Ser Gly Ser Gly Thr Pro Pro Thr Ser Ala Asn Ser Gln Tyr Phe 260 265 270 gat aat cat act atg act tca aat cga tca tca ctg ggt tca tca att 1160 Asp Asn His Thr Met Thr Ser Asn Arg Ser Ser Leu Gly Ser Ser Ile 275 280 285 290 tct att att gaa gct agt gtt gat tca tat caa tta gat cat ggt cga 1208 Ser Ile Ile Glu Ala Ser Val Asp Ser Tyr Gln Leu Asp His Gly Arg 295 300 305 tat caa tat tca ata act gct cga tta aat aat ggc aga ata aga tat 1256 Tyr Gln Tyr Ser Ile Thr Ala Arg Leu Asn Asn Gly Arg Ile Arg Tyr 310 315 320 tta tat cga tat tat caa gat ttt tat gat tta caa gtg aaa tta tta 1304 Leu Tyr Arg Tyr Tyr Gln Asp Phe Tyr Asp Leu Gln Val Lys Leu Leu 325 330 335 gaa tta ttt cct tat gaa gct ggg aga att gaa aat tct aaa aga ata 1352 Glu Leu Phe Pro Tyr Glu Ala Gly Arg Ile Glu Asn Ser Lys Arg Ile 340 345 350 att cca tct ata cca gga cct tta att aat gtc aat gat tca ata tca 1400 Ile Pro Ser Ile Pro Gly Pro Leu Ile Asn Val Asn Asp Ser Ile Ser 355 360 365 370 aaa tta cga aga gaa aaa ttg gat tat tat tta tca aat tta att gca 1448 Lys Leu Arg Arg Glu Lys Leu Asp Tyr Tyr Leu Ser Asn Leu Ile Ala 375 380 385 tta cct agt cat ata tct cga tca gaa gaa gta tta aaa tta ttt gat 1496 Leu Pro Ser His Ile Ser Arg Ser Glu Glu Val Leu Lys Leu Phe Asp 390 395 400 gtt tta gat aat gga ttt gat cga gaa act gat gct att aat aaa cga 1544 Val Leu Asp Asn Gly Phe Asp Arg Glu Thr Asp Ala Ile Asn Lys Arg 405 410 415 ttt tct aaa cca ata agt caa aaa tca aat tct cat caa gat aga tta 1592 Phe Ser Lys Pro Ile Ser Gln Lys Ser Asn Ser His Gln Asp Arg Leu 420 425 430 tct caa tat tcc aat ttt aac gtt tta caa caa caa caa caa caa cag 1640 Ser Gln Tyr Ser Asn Phe Asn Val Leu Gln Gln Gln Gln Gln Gln Gln 435 440 445 450 caa caa cag caa tat gct cat cat tca aga ggt tct gat aat tca cct 1688 Gln Gln Gln Gln Tyr Ala His His Ser Arg Gly Ser Asp Asn Ser Pro 455 460 465 act aat gaa tca tca ggt tca aat tta att aat tct tct tct cat aat 1736 Thr Asn Glu Ser Ser Gly Ser Asn Leu Ile Asn Ser Ser Ser His Asn 470 475 480 gat tca tca tta tct tca tca cca cca cca cca cca cca caa act gtc 1784 Asp Ser Ser Leu Ser Ser Ser Pro Pro Pro Pro Pro Pro Gln Thr Val 485 490 495 acc acc acg aac acc acg aac acc acc ata acc aca gac tcc tca tca 1832 Thr Thr Thr Asn Thr Thr Asn Thr Thr Ile Thr Thr Asp Ser Ser Ser 500 505 510 aaa caa cca aaa gcc aaa gtg aaa ttt tat ttt gat gat gat ata ttt 1880 Lys Gln Pro Lys Ala Lys Val Lys Phe Tyr Phe Asp Asp Asp Ile Phe 515 520 525 530 gta tta tta atc cca acc aat tta cga tta caa gat tta aaa tca aaa 1928 Val Leu Leu Ile Pro Thr Asn Leu Arg Leu Gln Asp Leu Lys Ser Lys 535 540 545 tta ttt aaa cga tta gaa ttg gat att act tat aaa tat gaa aaa cct 1976 Leu Phe Lys Arg Leu Glu Leu Asp Ile Thr Tyr Lys Tyr Glu Lys Pro 550 555 560 gat caa caa caa aaa cct aca tca gaa tca att cat tta ttt ttg aaa 2024 Asp Gln Gln Gln Lys Pro Thr Ser Glu Ser Ile His Leu Phe Leu Lys 565 570 575 aat gat ttt gaa gat ttt tta att gaa aat gaa act agc aac aac aac 2072 Asn Asp Phe Glu Asp Phe Leu Ile Glu Asn Glu Thr Ser Asn Asn Asn 580 585 590 aat ctg gaa att gat ttc gaa aat gaa att att aaa gaa aaa tta gga 2120 Asn Leu Glu Ile Asp Phe Glu Asn Glu Ile Ile Lys Glu Lys Leu Gly 595 600 605 610 gaa ttt gaa gtt aat gat gat gaa aaa ttt caa agt att tta ttt gat 2168 Glu Phe Glu Val Asn Asp Asp Glu Lys Phe Gln Ser Ile Leu Phe Asp 615 620 625 aaa tgt aaa tta atg gtt tta gta tat taaacagaga tcaataagag 2215 Lys Cys Lys Leu Met Val Leu Val Tyr 630 635 agagagagag agacat 2231 12 635 PRT Candida albicans 12 Met Ile Lys Thr Phe Arg Lys Ser Lys Arg Leu Ser Ser Asn Ser Ser 1 5 10 15 Ser Pro Lys Lys Thr Ile Ser Arg Val Ser Ser Thr Ser Ser Asn Gln 20 25 30 Thr Ser His Asp Gly Ile Leu Gln Ser Pro Lys Lys Val Ile Arg Ala 35 40 45 Leu Tyr Asp Tyr Glu Pro Gln Gly Pro Gly Glu Leu Lys Phe Phe Lys 50 55 60 Gly Asp Phe Phe His Val Leu Asn Asp Val Asp Asp Glu Leu His Lys 65 70 75 80 Glu Ala Glu Arg Asn Gly Trp Ile Glu Ala Thr Asn Pro Met Thr Gln 85 90 95 Leu Lys Gly Met Val Pro Ile Ser Tyr Phe Glu Ile Phe Asp Arg Ser 100 105 110 Arg Pro Thr Val Thr Ala Ser Ser Asn Ser Phe Thr Asn Ser Ile Asp 115 120 125 Ile Gln His Gln His Gln Gln Gly Ile His Asn Gly Thr Gly Asn Arg 130 135 140 Asn Leu Asn Gln Thr Leu Tyr Ala Val Thr Leu Tyr Glu Phe Lys Ala 145 150 155 160 Glu Arg Asp Asp Glu Leu Asp Ile Met Pro Asn Glu Asn Leu Ile Ile 165 170 175 Cys Ala His His Asp Tyr Glu Trp Phe Ile Ala Lys Pro Ile Asn Arg 180 185 190 Leu Gly Gly Pro Gly Leu Val Pro Val Ser Tyr Val Lys Ile Ile Asp 195 200 205 Leu Leu Asn Pro Asn Ser His Tyr Thr Ser Ile Asp Thr Ser Arg Arg 210 215 220 Ser Gln Val Ile Gln Val Ile Asn Gly Phe Asn Ile Pro Thr Val Glu 225 230 235 240 Gln Trp Lys Asn Gln Thr Ala Lys Tyr Gln Ala Ser Thr Ile Pro Leu 245 250 255 Gly Ser Ile Ser Gly Ser Gly Thr Pro Pro Thr Ser Ala Asn Ser Gln 260 265 270 Tyr Phe Asp Asn His Thr Met Thr Ser Asn Arg Ser Ser Leu Gly Ser 275 280 285 Ser Ile Ser Ile Ile Glu Ala Ser Val Asp Ser Tyr Gln Leu Asp His 290 295 300 Gly Arg Tyr Gln Tyr Ser Ile Thr Ala Arg Leu Asn Asn Gly Arg Ile 305 310 315 320 Arg Tyr Leu Tyr Arg Tyr Tyr Gln Asp Phe Tyr Asp Leu Gln Val Lys 325 330 335 Leu Leu Glu Leu Phe Pro Tyr Glu Ala Gly Arg Ile Glu Asn Ser Lys 340 345 350 Arg Ile Ile Pro Ser Ile Pro Gly Pro Leu Ile Asn Val Asn Asp Ser 355 360 365 Ile Ser Lys Leu Arg Arg Glu Lys Leu Asp Tyr Tyr Leu Ser Asn Leu 370 375 380 Ile Ala Leu Pro Ser His Ile Ser Arg Ser Glu Glu Val Leu Lys Leu 385 390 395 400 Phe Asp Val Leu Asp Asn Gly Phe Asp Arg Glu Thr Asp Ala Ile Asn 405 410 415 Lys Arg Phe Ser Lys Pro Ile Ser Gln Lys Ser Asn Ser His Gln Asp 420 425 430 Arg Leu Ser Gln Tyr Ser Asn Phe Asn Val Leu Gln Gln Gln Gln Gln 435 440 445 Gln Gln Gln Gln Gln Gln Tyr Ala His His Ser Arg Gly Ser Asp Asn 450 455 460 Ser Pro Thr Asn Glu Ser Ser Gly Ser Asn Leu Ile Asn Ser Ser Ser 465 470 475 480 His Asn Asp Ser Ser Leu Ser Ser Ser Pro Pro Pro Pro Pro Pro Gln 485 490 495 Thr Val Thr Thr Thr Asn Thr Thr Asn Thr Thr Ile Thr Thr Asp Ser 500 505 510 Ser Ser Lys Gln Pro Lys Ala Lys Val Lys Phe Tyr Phe Asp Asp Asp 515 520 525 Ile Phe Val Leu Leu Ile Pro Thr Asn Leu Arg Leu Gln Asp Leu Lys 530 535 540 Ser Lys Leu Phe Lys Arg Leu Glu Leu Asp Ile Thr Tyr Lys Tyr Glu 545 550 555 560 Lys Pro Asp Gln Gln Gln Lys Pro Thr Ser Glu Ser Ile His Leu Phe 565 570 575 Leu Lys Asn Asp Phe Glu Asp Phe Leu Ile Glu Asn Glu Thr Ser Asn 580 585 590 Asn Asn Asn Leu Glu Ile Asp Phe Glu Asn Glu Ile Ile Lys Glu Lys 595 600 605 Leu Gly Glu Phe Glu Val Asn Asp Asp Glu Lys Phe Gln Ser Ile Leu 610 615 620 Phe Asp Lys Cys Lys Leu Met Val Leu Val Tyr 625 630 635 

We claim:
 1. An isolated gene encoding a protein of Candida albicans selected from the group consisting of CaCla4p (SEQ ID NO:8), Cst20p (SEQ ID NO: 6), CaCdc42p (SEQ ID NO: 10) and CaBem1p (SEQ ID NO: 12).
 2. An isolated Candida albicans protein selected from the group consisting of CaCla4p (SEQ ID NO:8), Cst20p (SEQ ID NO: 6), CaCdc42p (SEQ ID NO: 10) and CaBem1p (SEQ ID NO: 12).
 3. An in vitro screening assay for identifying a potential anti-fungal compound, said assay comprising the steps of: a) combining at least one protein selected from the group consisting of CaCla4p (SEQ ID NO:8), Cst20p (SEQ ID NO: 6), CaCdc42p (SEQ ID NO: 10) and CaBem1p (SEQ ID NO: 12) with a target that interacts with said protein in a protein/target interaction; b) adding a test compound to the protein/target mixture; and c) measuring the protein/target interaction, wherein an interruption of the protein/target interaction indicates that said test compound is a potential inhibitor.
 4. An assay as defined in claim 3, wherein said at least one protein is CaCdc42p, and the target is Cacla4p.
 5. An assay as defined in claim 3, wherein said protein is CaCdc42p and the target is Cst20p.
 6. An assay as defined in claim 3, wherein said protein is CaCla4p and the target is CaBem1p.
 7. An assay as defined in claim 3, wherein said protein is CaBem1p and the target is Cst20p. 